Automated instrumentation for production of T-cell receptor peptide libraries

ABSTRACT

The present disclosure provides instrumentation and automated methods for creating cell surface display libraries, where the cells of the library display engineered peptides on their cell surfaces for identification of antigens that bind to T-cell receptors. The engineered peptides may be putative antigens or binding regions of the T-cell receptors.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 17/690,641, filed 9Mar. 2022, which is a continuation of U.S. Ser. No. 17/016,454, filed 10Sep. 2020, now U.S. Pat. No. 11,293,117; which is a continuation of U.S.Ser. No. 16/904,640, filed 18 Jun. 2020, U.S. Pat. No. 10,774,446; whichis a continuation of U.S. Ser. No. 16/827,164, filed 23 Mar. 2020, nowU.S. Pat. No. 10,711,374; which is a continuation of U.S. Ser. No.16/670,340, filed 31 Oct. 2019, now U.S. Pat. No. 10,676,842; which is acontinuation of U.S. Ser. No. 16/392,605, filed Apr. 23, 2019, now U.S.Pat. No. 10,557,216 which claims priority to U.S. Provisional PatentApplication Ser. No. 62/671,266, entitled “MULTIPLEXED METHODS FORPRODUCTION AND USE OF CELL SURFACE DISPLAY LIBRARIES,” filed May 14,2018; and U.S. Patent Application Ser. No. 62/662,126, entitled“MULTIPLEXED METHODS FOR PRODUCTION AND USE OF CELL SURFACE DISPLAYLIBRARIES,” filed Apr. 24, 2018, both of which are hereby incorporatedby reference in their entireties for all purposes.

FIELD OF THE INVENTION

The present disclosure relates to automated multi-module instruments andmultiplexed methods of making cell surface display libraries usinggenomic editing technologies.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

The binding and activation of a T-cell receptor (TCR) to its specificantigen has been difficult to identify in high throughput systems due tothe diversity of major histocompatibility complexes, the variety ofpotential antigens, and the diversity of T-cells in humans and animals.Conventional techniques such as HPLC require a priori information aboutthe TCR target, and the identification process can be both lengthy andcumbersome.

There is thus a need in the art for better and more robust means foridentifying specific antigens for TCRs in a high throughput, multiplexedmanner. The present invention addresses this need.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present disclosure provides compositions, instruments and automatedmethods for providing multiplexed displays of engineered peptides on thesurface of a population of cells. The engineered peptides are preferablyexpressed in the cells under conditions that provide both secretion anddisplay of the engineered peptides on the cell surfaces, thus providingaccess of the engineered peptides to potential binding targets. The cellpopulations can be engineered using an automated editing system thatprovides for one or more targeted edits per cell, allowing for therational design of a library of cells having engineered peptidesdisplayed on their respective surfaces. Accordingly, this disclosuredescribes various automated methods for expressing and displayingengineered peptides on cells.

In some embodiments, the disclosure provides a method of producing acell library expressing engineered peptides for identification of T-cellreceptor (TCR)-antigen binding, the method comprising providing apopulation of cells, processing the population of cells using aninstrument for multiplexed nuclease-directed genome editing usingintroduced nucleic acids and a nucleic acid-directed nuclease to createcells comprising nucleic acids that encode engineered peptidesconfigured to be displayed on a surface of the cells, incubating theprocessed cells to facilitate nucleic acid editing in the cells, whereinthe editing provides nucleic acids that encode engineered peptideantigens in the cells, and allowing the cells to express and display theengineered peptides on the surface of the cells.

In some aspects, the engineered peptides are putative TCR bindingantigens. In other aspects, the engineered peptides comprise predictedTCR binding regions. In some aspects the engineered peptides derive froma target genomic sequence and contain an inserted N-terminus orC-terminus cell surface display conferring tag.

In other embodiments, the disclosure provides methods of producing acell library expressing engineered putative T-cell receptor (TCR)antigens on the surface of the cells, the method comprising providing apopulation of cells, processing the population of cells using aninstrument for multiplexed nuclease-directed genome editing usingintroduced nucleic acids and a nuclease to create cells comprisingnucleic acids that encode engineered peptide antigens configured to bedisplayed on a surface of the cells, incubating the processed cells tofacilitate nucleic acid editing in the cells, wherein the editingprovides nucleic acids that encode engineered peptide antigens in thecells, and allowing the cells to express and display the engineeredpeptide antigens that are putative TCR antigens on the surface of thecells.

The engineered peptide antigens in the population of edited cellspreferably comprise rationally designed peptides that can be displayedon a cell surface in a manner by which the antigen is available forbinding to a TCR target, either known TCRs and/or orphan TCRs. In someaspects, the engineered peptide antigens are known antigens of one ormore TCRs.

In specific embodiments the antigen is displayed on the cell surface aspart of an MHC (e.g. HLA) which includes the peptide antigen therebyforming a TCR ligand. Accordingly, in some aspects, the cells displaythe engineered peptide antigens as part of a ligand. In some aspects,the cells co-express putative TCR antigens and MHC molecules.

Peptide antigens for use with the systems and methods of the disclosureinclude known antigens of one or more TCRs, predicted antigens for oneor more TCRs, or random peptides created using nucleases in theautomated cell editing instruments of the present disclosure. Inembodiments, the peptides that are displayed are created using forwardengineering to create peptide sequences based on predictions of whatantigens may be useful for specific TCRs.

In some embodiments, the disclosure provides methods of producing a celllibrary expressing engineered peptides derived from the cells' genome(s)on the surface of cells, the method comprising providing a population ofcells, processing the population of cells using an instrument formultiplexed nuclease-directed genome editing using introduced nucleicacids and a nuclease to create cells comprising nucleic acids thatencode engineered proteins configured with an N-terminus or C-terminuscell surface display conferring tag to be displayed on a surface of thecells, incubating the processed cells to facilitate nucleic acid editingin the cells, wherein the editing provides nucleic acids that encodecell surface display conferring tags at the N-terminus or C-terminus ofengineered proteins in the cells, and allowing the cells to express anddisplay the engineered proteins on the surface of the cell.

In some embodiments, the disclosure provides multiplexed method foridentifying peptides that selectively bind one or more TCRs, the methodcomprising providing a population of cells, processing the population ofcells using an automated system for multiplexed nuclease-directed genomeediting, wherein the system comprises the steps of introducing nucleicacids that encode engineered peptide antigens and a nuclease to apopulation of cells, incubating the cells to facilitate nucleic acidediting in the cells, allowing the edited cells to express and displaythe engineered peptide antigens on the surface of the edited cells,screening the edited cells displaying the engineered peptide antigensagainst one or more TCRs, and identifying the edited cells expressingengineered peptide antigens that selectively bind to one or more TCRs.

In some aspects, the disclosure further provides isolating the nucleicacids encoding the engineered peptide antigens that selectively bind toone or more TCRs from the cells. In some aspects, the disclosure furtherprovides isolating the nucleic acids encoding the engineered peptidesthat selectively bind to one or more putative TCR antigens from thecells.

In some aspects, the cells encoding specific peptides are identified bydetection of a barcode associated with the engineered peptides. In someaspects, the cells encoding specific are identified by detection of abarcode associated with the engineered peptide antigens that selectivelybind to one or more TCRs. In some embodiments, the barcode is used toisolate and/or further identify or process the cells and nucleic acidsencoding the peptides for further analysis. In such embodiments, thebarcode can be used as a “handle” to pull out the cells of interest forfurther analysis.

In specific aspects, the disclosure provides a method of producing acell library expressing engineered peptide antigens on the surface ofcells by providing a population of cells, editing the population ofcells using one or more introduced nucleic acids comprising a guide RNAcovalently linked to a donor DNA (e.g., homology arm) that selectivelybinds to a genomic region of interest and a nuclease, incubating thecells to facilitate nucleic acid editing in the cells, wherein theediting provides nucleic acids that encode engineered peptide antigensin the cells, and allowing the cells to express and display theengineered peptide antigens on the surface of the edited cells.

In other specific aspects, the disclosure provides a method of producinga cell library expressing engineered peptide antigens on the surface ofcells by providing a population of cells, editing the population ofcells employing an automated instrument for multiplexednuclease-directed genome editing using introduced nucleic acidscomprising the edits and a nuclease, incubating the cells to facilitatenucleic acid editing in the cells, wherein the editing provides nucleicacids that encode engineered peptide antigens in the cells, and allowingthe cells to express and display the engineered peptide antigens on thesurface of the edited cells.

The engineered peptide antigens in the population of edited cellspreferably comprise rationally designed peptides that can be displayedon a cell surface in a manner by which the antigen is available forbinding to a T-cell receptor (“TCR”) target. In some aspects of thedisclosure, the engineered peptides are derived from target genomicsequences.

Various nucleases may be used with the editing methods of the presentdisclosure, including zinc finger nucleases, meganucleases, TALENS, andnucleic acid-directed nucleases (e.g., RNA-directed nucleases).Preferably, the editing methods are carried out using nucleicacid-directed nucleases, and more preferably RNA-directed nucleases.

In specific embodiments, the disclosure provides multiplexed methods foridentifying cells expressing engineered putative TCR antigens on theirsurface comprising providing a population of cells, editing thepopulation of cells using an automated instrument for multiplexednuclease-directed genome editing and introduced nucleic acids and anuclease to create nucleic acids that encode putative TCR antigens inthe cells, incubating the cells to facilitate nucleic acid editing inthe cells, allowing the cells to express and display the engineeredputative TCR antigens on their surface, screening the cells displayingthe engineered putative TCR antigens against a target, and identifyingthe cells expressing engineered putative TCR antigens that selectivelybind to the target.

In one embodiment, the disclosure provides multiplexed methods foridentifying cells expressing engineered putative TCR antigens on theirsurface comprising providing a population of cells; editing thepopulation of cells using an automated instrument for multiplexednuclease-directed genome editing and introduced nucleic acids and anucleic acid-directed nuclease thereby creating cells comprising nucleicacids that encode engineered putative TCR antigens, incubating the cellsto facilitate nucleic acid editing in the cells, allowing the editedcells to express and display the engineered putative TCR antigens ontheir surface, screening the cells displaying the engineered putativeTCR antigens against a target, selecting the cells expressing engineeredputative TCR antigens that selectively bind to one or more TCR targets,and detecting or isolating the nucleic acid encoding the antigens.Alternatively, the conditions can be varied to determine the selectivityunder different conditions.

Detection of a specific peptide in a cell of interest can beaccomplished using various methods known in the art, e.g., sequencing,hybridization, identification of a barcode indicative of an antigensequence, and the like. Barcodes and other features can also be used forfurther analysis, e.g., by providing a basis for identifying and/orisolating cells of interest encoding peptides identified for elucidationof TCR binding.

In one aspect, the disclosure provides methods for the immobilization ofone or more engineered peptide antigens on a cell surface by providingfusion proteins for display of one or more engineered peptide antigenson a yeast cell surface. In one embodiment, the disclosure provides formethods for displaying an engineered peptide antigen as part of an MHCantigen (e.g., HLA) on the cell surface. In certain embodiments, thecells display multiple copies of a single engineered antigen.

In specific embodiments, the disclosure provides methods for providingreceptors or binding regions thereof on the cell

In specific embodiments, the disclosure provides multiplexed methods foridentifying cells expressing engineered predicted TCR binding regions(e.g., predicted binding regions of orphan TCRs) on their surfacecomprising providing a population of cells, editing the population ofcells using an automated instrument for multiplexed nuclease-directedgenome editing and introduced nucleic acids and a nuclease to createnucleic acids that encode TCR binding regions in the cells, incubatingthe cells to facilitate nucleic acid editing in the cells, allowing thecells to express and display the engineered TCR binding regions on theirsurface, screening the cells displaying the engineered TCR bindingregions against a target, and identifying the cells expressingengineered TCR binding regions that selectively bind to putativeantigens.

In one embodiment, the disclosure provides multiplexed methods foridentifying cells expressing engineered predicted binding regions fromTCRs (e.g., orphan TCRs) on their surface comprising: providing apopulation of cells, editing the population of cells using an automatedinstrument for multiplexed nuclease-directed genome editing andintroduced nucleic acids and a nucleic acid-directed nuclease therebycreating cells comprising nucleic acids that encode engineered TCRbinding regions, incubating the cells to facilitate nucleic acid editingin the cells, allowing the edited cells to express and display theengineered TCR binding regions on their surface, screening the cellsdisplaying the engineered TCR binding regions against a target, andidentifying the cells expressing engineered TCR binding regions thatselectively bind to one or more putative TCR binding antigens.Alternatively, the conditions can be varied to determine the selectivityunder different conditions.

These aspects and other features and advantages of the invention aredescribed below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings in which:

FIG. 1 is a schematic showing the structure of the TCRα and TCRβ loci.

FIG. 2 is a schematic showing how TCR gene segments rearrange duringT-cell development to form complete V-domain exons.

FIG. 3 is a schematic showing the cluster of gene segments encoding theδ chain within the TCRα locus.

FIGS. 4A-4D depict an automated multi-module instrument and componentsthereof with which to generate the cell surface libraries of thedisclosure.

FIG. 5A depicts one embodiment of a rotating growth vial for use withthe cell growth module described herein. FIG. 5B illustrates aperspective view of one embodiment of a rotating growth vial in a cellgrowth module. FIG. 5C depicts a cut-away view of the cell growth modulefrom FIG. 5B. FIG. 5D illustrates the cell growth module of FIG. 5Bcoupled to LED, detector, and temperature regulating components.

FIG. 6A is a model of tangential flow filtration used in the TFF devicepresented herein. FIG. 6B depicts a top view of a lower member of oneembodiment of an exemplary TFF device. FIG. 6C depicts a top view ofupper and lower members and a membrane of an exemplary TFF device. FIG.6D depicts a bottom view of upper and lower members and a membrane of anexemplary TFF device. FIGS. 6E-6I depict various views of an embodimentof a TFF module comprising a TFF device and having fluidically coupledreservoirs for retentate, filtrate, and exchange buffer.

FIGS. 7A and 7B are top perspective and bottom perspective views,respectively, of flow-through electroporation devices (here, there aresix such devices co-joined). FIG. 7C is a top view of one embodiment ofan exemplary flow-through electroporation device. FIG. 7D depicts a topview of a cross section of the electroporation device of FIG. 7C. FIG.7E is a side view cross section of a lower portion of theelectroporation devices of FIGS. 7C and 7D.

FIG. 8A depicts a simplified graphic of a workflow for singulating,editing and normalizing cells after nucleic acid-guided nuclease genomeediting in a solid wall device. FIG. 8B is a photograph of oneembodiment of a solid wall device. FIGS. 8C-8E are photographs of E.coli cells singulated (via Poisson distribution) and grown into coloniesin microwells in a solid wall device with a permeable bottom at low,medium, and high magnification, respectively. FIG. 8F is a simplifiedblock diagram of methods for enriching for live cells that have beenedited via nucleic acid-guided nuclease editing that do not involvesingulation or a singulation device and instead utilize cell growth inliquid and induction of editing. FIG. 8G depicts a typical growth curvefor cells in culture. FIG. 8H is a graphic depiction of methods forgrowing, inducing, editing, enriching, and screening for edited cells ina population of cells.

FIGS. 9A and 9B depict an example reagent cartridge for use in anautomated multi-module cell editing instrument.

FIG. 10 is a flow chart of an example method for automated multi-modulecell editing to produce the cell libraries as described herein.

FIG. 11 is a simplified flow chart of two exemplary methods (1100 a and1100 b) that may be performed by an automated multi-module cell editinginstrument comprising a singulation device.

FIG. 12 is a simplified block diagram of an embodiment of an exemplaryautomated multi-module cell processing instrument comprising a solidwall singulation/growth/editing/normalization module.

FIG. 13 is a simplified block diagram of an alternative embodiment of anexemplary automated multi-module cell processing instrument comprising asolid wall singulation/growth/editing/normalization module.

FIG. 14 is a simplified process diagram of an embodiment of an exemplaryautomated multi-module cell processing instrument.

FIG. 15 is a graph demonstrating the effectiveness of a 2-paddlerotating growth vial and cell growth device as described herein forgrowing an EC23 cell culture vs. a conventional cell shaker.

FIG. 16 is a graph demonstrating the effectiveness of a 3-paddlerotating growth vial and cell growth device as described herein forgrowing an EC23 cell culture vs. a conventional cell shaker.

FIG. 17 is a graph demonstrating the effectiveness of a 4-paddlerotating growth vial and cell growth device as described herein forgrowing an EC138 cell culture vs. a conventional orbital cell shaker.

FIG. 18 is a graph demonstrating the effectiveness of a 2-paddlerotating growth vial and cell growth device as described herein forgrowing an EC138 cell culture vs. a conventional orbital cell shaker.

FIG. 19 is a graph demonstrating real-time monitoring of growth of anEC138 cell culture to OD₆₀₀ employing the cell growth device asdescribed herein where a 2-paddle rotating growth vial was used.

FIG. 20 is a graph demonstrating real-time monitoring of growth of s288cyeast cell culture OD₆₀₀ employing the cell growth device as describedherein where a 2-paddle rotating growth vial was used.

FIG. 21A is a graph plotting filtrate conductivity against filterprocessing time for an E. coli culture processed in the cellconcentration device/module described herein.

FIG. 21B is a graph plotting filtrate conductivity against filterprocessing time for a yeast culture processed in the cell concentrationdevice/module described herein.

FIG. 22A is a bar graph showing the results of electroporation of E.coli using a device of the disclosure and a comparator electroporationdevice. FIG. 22B is a bar graph showing uptake, cutting, and editingefficiencies of E. coli cells transformed via an FTEP as describedherein benchmarked against a comparator electroporation device.

FIG. 23 is a bar graph showing the results of electroporation of S.cerevisiae using an FTEP device of the disclosure and a comparatorelectroporation method.

It should be understood that the drawings are not necessarily to scale,and that like reference numbers refer to like features.

DETAILED DESCRIPTION

All of the functionalities described in connection with one embodimentof the methods, devices or instruments described herein are intended tobe applicable to the additional embodiments of the methods, devices andinstruments described herein except where expressly stated or where thefeature or function is incompatible with the additional embodiments. Forexample, where a given feature or function is expressly described inconnection with one embodiment but not expressly mentioned in connectionwith an alternative embodiment, it should be understood that the featureor function may be deployed, utilized, or implemented in connection withthe alternative embodiment unless the feature or function isincompatible with the alternative embodiment.

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions ofmolecular biology (including recombinant techniques), cell biology,biochemistry, and genetic engineering technology, which are within theskill of those who practice in the art. Such conventional techniques anddescriptions can be found in standard laboratory manuals such as Greenand Sambrook, Molecular Cloning: A Laboratory Manual. 4th, ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2014);Current Protocols in Molecular Biology, Ausubel, et al. eds., (2017);Neumann, et al., Electroporation and Electrofusion in Cell Biology,Plenum Press, New York, 1989; and Chang, et al., Guide toElectroporation and Electrofusion, Academic Press, California (1992),all of which are herein incorporated in their entirety by reference forall purposes. Nucleic acid-guided nuclease techniques can be found in,e.g., Genome Editing and Engineering from TALENs and CRISPRs toMolecular Surgery, Appasani and Church (2018); and CRISPR: Methods andProtocols, Lindgren and Charpentier (2015); both of which are hereinincorporated in their entirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” refers toone or more cells, and reference to “the system” includes reference toequivalent steps, methods and devices known to those skilled in the art,and so forth. Additionally, it is to be understood that terms such as“left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,”“length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,”“outer” that may be used herein merely describe points of reference anddo not necessarily limit embodiments of the present disclosure to anyparticular orientation or configuration. Furthermore, terms such as“first,” “second,” “third,” etc., merely identify one of a number ofportions, components, steps, operations, functions, and/or points ofreference as disclosed herein, and likewise do not necessarily limitembodiments of the present disclosure to any particular configuration ororientation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, formulations and methodologies that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in smaller ranges, and arealso encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, features and procedures well known to thoseskilled in the art have not been described in order to avoid obscuringthe invention. The terms used herein are intended to have the plain andordinary meaning as understood by those of ordinary skill in the art.

The term “complementary” as used herein refers to Watson-Crick basepairing between nucleotides and specifically refers to nucleotideshydrogen bonded to one another with thymine or uracil residues linked toadenine residues by two hydrogen bonds and cytosine and guanine residueslinked by three hydrogen bonds. In general, a nucleic acid includes anucleotide sequence described as having a “percent complementarity” or“percent homology” to a specified second nucleotide sequence. Forexample, a nucleotide sequence may have 80%, 90%, or 100%complementarity to a specified second nucleotide sequence, indicatingthat 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence arecomplementary to the specified second nucleotide sequence. For instance,the nucleotide sequence 3′-TCGA-5′ is 100% complementary to thenucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-TCGA-5′is 100% complementary to a region of the nucleotide sequence5′-TTAGCTGG-3′.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites, nuclear localization sequences, enhancers, and the like,which collectively provide for the replication, transcription andtranslation of a coding sequence in a recipient cell. Not all of thesetypes of control sequences need to be present so long as a selectedcoding sequence is capable of being replicated, transcribed and—for somecomponents—translated in an appropriate host cell.

As used herein the term “donor DNA” or “donor nucleic acid” refers tonucleic acid that is designed to introduce a DNA sequence modification(insertion, deletion, substitution) into a locus by homologousrecombination using nucleic acid-guided nucleases. For homology-directedrepair, the donor DNA must have sufficient homology to the regionsflanking the “cut site” or site to be edited in the genomic targetsequence. The length of the homology arm(s) will depend on, e.g., thetype and size of the modification being made. In many instances andpreferably, the donor DNA will have two regions of sequence homology(e.g., two homology arms) to the genomic target locus. Preferably, an“insert” region or “DNA sequence modification” region—the nucleic acidmodification that one desires to be introduced into a genome targetlocus in a cell-will be located between two regions of homology. The DNAsequence modification may change one or more bases of the target genomicDNA sequence at one specific site or multiple specific sites. A changemay include changing 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, 150, 200, 300, 400, or 500 or more base pairs of the targetsequence. A deletion or insertion may be a deletion or insertion of 1,2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, 400, or500 or more base pairs of the target sequence.

The term “engineered peptide antigen” encompasses naturally occurringand synthetic polypeptides and protein constructs that comprise asynthetic polypeptide or naturally occurring peptide associated withdifferent elements, like, for instance, peptides for MHC display of thepeptide, an immobilization peptide, reporter peptide or secretionpeptide. engineered peptide antigens are encoded and/or expressed from arecombinant nucleic acid that may be engineered to include sequencevariants, recombinant promoters, transcriptional control elements,fusion peptides, other modifications, or any combination of two or morethereof. The peptide presentation may include presentation of all or aportion of a protein of interest. In some embodiments, engineeredpeptide antigens comprise a binding motif that is modified by a couplingenzyme, resulting in the coupling of a second binding target to thebinding motif. In some embodiments, the second binding target is coupledto the engineered peptide antigen intracellularly.

As used herein, “enrichment” refers to enriching for edited cells bysingulation, optionally inducing editing, and growth of singulated cellsinto terminal-sized colonies (e.g., saturation or normalization ofcolony growth).

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to apolynucleotide comprising 1) a guide sequence capable of hybridizing toa genomic target locus, and 2) a scaffold sequence capable ofinteracting or complexing with a nucleic acid-guided nuclease.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or, more often in the context of the presentdisclosure, between two nucleic acid molecules. The term “homologousregion” or “homology arm” refers to a region on the donor DNA with acertain degree of homology with the target genomic DNA sequence.Homology can be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When a position in thecompared sequence is occupied by the same base or amino acid, then themolecules are homologous at that position. A degree of homology betweensequences is a function of the number of matching or homologouspositions shared by the sequences.

As used herein, the terms “leader peptide”, “secretion peptide” orsecretion leader peptide refers to any signaling sequence that directs asynthesized fusion protein away from the translation site, includingsignaling sequences that will result in the fusion peptide crossing thecell membrane and being secreted.

“Operably linked” refers to an arrangement of elements where thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the transcription, and in some cases, thetranslation, of a coding sequence. The control sequences need not becontiguous with the coding sequence so long as they function to directthe expression of the coding sequence. Thus, for example, interveninguntranslated yet transcribed sequences can be present between a promotersequence and the coding sequence and the promoter sequence can still beconsidered “operably linked” to the coding sequence. In fact, suchsequences need not reside on the same contiguous DNA molecule (i.e.chromosome) and may still have interactions resulting in alteredregulation.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably. Proteins may or may not be made up entirely of aminoacids.

A “promoter” or “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase and initiating transcription of apolynucleotide or polypeptide coding sequence such as messenger RNA,ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind ofRNA transcribed by any class of any RNA polymerase I, II or III.Promoters may be constitutive or inducible, and in someembodiments—particularly many embodiments in which selection isemployed—the transcription of at least one component of the nucleicacid-guided nuclease editing system is under the control of an induciblepromoter.

As used herein the term “selectable marker” refers to a gene introducedinto a cell, which confers a trait suitable for artificial selection.General use selectable markers are well-known to those of ordinary skillin the art. Drug selectable markers such as ampicillin/carbenicillin,kanamycin, chloramphenicol, erythromycin, tetracycline, gentamicin,bleomycin, streptomycin, rifampicin, puromycin, hygromycin, blasticidin,and G418 may be employed. In other embodiments, selectable markersinclude, but are not limited to sugars such as rhamnose. human nervegrowth factor receptor (detected with a MAb, such as described in U.S.Pat. No. 6,365,373); truncated human growth factor receptor (detectedwith MAb); mutant human dihydrofolate reductase (DHFR; fluorescent MTXsubstrate available); secreted alkaline phosphatase (SEAP; fluorescentsubstrate available); human thymidylate synthase (TS; confers resistanceto anti-cancer agent fluorodeoxyuridine); human glutathioneS-transferase alpha (GSTA1; conjugates glutathione to the stem cellselective alkylator busulfan; chemoprotective selectable marker in CD34+cells); CD24 cell surface antigen in hematopoietic stem cells; human CADgene to confer resistance to N-phosphonacetyl-L-aspartate (PALA); humanmulti-drug resistance-1 (MDR-1; P-glycoprotein surface proteinselectable by increased drug resistance or enriched by FACS); human CD25(IL-2a; detectable by Mab-FITC); Methylguanine-DNA methyltransferase(MGMT; selectable by carmustine); and Cytidine deaminase (CD; selectableby Ara-C). “Selective medium” as used herein refers to cell growthmedium to which has been added a chemical compound or biological moietythat selects for or against selectable markers.

The term “specifically binds” as used herein includes an interactionbetween two molecules, e.g., an engineered peptide antigen and a bindingtarget, with a binding affinity represented by a dissociation constantof about 10⁻⁷M, about 10⁻⁸M, about 10⁻⁹ M, about 10⁻¹⁰ M, about 10⁻¹¹M,about 10⁻¹²M, about 10⁻¹³M, about 10⁻¹⁴M or about 10⁻¹⁵M.

The terms “target genomic DNA sequence”, “target sequence”, or “genomictarget locus” refer to any locus in vitro or in vivo, or in a nucleicacid (e.g., genome) of a cell or population of cells, in which a changeof at least one nucleotide is desired using a nucleic acid-guidednuclease editing system. The target sequence can be a genomic locus orextrachromosomal locus.

The term “variant” may refer to a polypeptide or polynucleotide thatdiffers from a reference polypeptide or polynucleotide, but retainsessential properties. A typical variant of a polypeptide differs inamino acid sequence from another reference polypeptide. Generally,differences are limited so that the sequences of the referencepolypeptide and the variant are closely similar overall and, in manyregions, identical. A variant and reference polypeptide may differ inamino acid sequence by one or more modifications (e.g., substitutions,additions, and/or deletions). A variant of a polypeptide may be aconservatively modified variant. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code (e.g., anon-natural amino acid). A variant of a polypeptide may be naturallyoccurring, such as an allelic variant, or it may be a variant that isnot known to occur naturally.

A “vector” is any of a variety of nucleic acids that comprise a desiredsequence or sequences to be delivered to and/or expressed in a cell.Vectors are typically composed of DNA, although RNA vectors are alsoavailable. Vectors include, but are not limited to, plasmids, fosmids,phagemids, virus genomes, synthetic chromosomes, and the like. As usedherein, the phrase “engine vector” comprises a coding sequence for anuclease to be used in the nucleic acid-guided nuclease systems andmethods of the present disclosure. The engine vector may also comprise,in a bacterial system, the λ Red recombineering system or an equivalentthereto. Engine vectors also typically comprise a selectable marker. Asused herein the phrase “editing vector” comprises a donor nucleic acid,optionally including an alteration to the target sequence that preventsnuclease binding at a PAM or spacer in the target sequence after editinghas taken place, and a coding sequence for a gRNA. The editing vectormay also comprise a selectable marker and/or a barcode. In someembodiments, the engine vector and editing vector may be combined; thatis, all editing and selection components may be found on a singlevector. Further, the engine and editing vectors comprise controlsequences operably linked to, e.g., the nuclease coding sequence,recombineering system coding sequences (if present), donor nucleic acid,guide nucleic acid, and selectable marker(s).

Cell Libraries, Screening and Editing Methods

The present disclosure provides multiplexed methods and automatedinstruments for creating cell populations with cell surface displayswhere the methods employ editing technologies. The cell populationsedited using the multiplexed and automated instrumentation of thedisclosure comprise one or more putative receptor antigens displayed ona cell's surface and available for binding to a binding target. Thecells that may be edited and used according to the disclosure include,but are not limited to, bacterial cells, yeast cells and mammaliancells. In addition, the cells that are edited may include sequences thatare heterologous to the host (e.g., editing of mammalian sequencesinserted into a yeast or bacterial genome).

In particular the methods and automated instruments used to create thecells are useful in identifying antigens that specifically bind toT-cell receptors (TCRs). The ability to quickly and easily identifyantigens, e.g. putative antigen targets of orphan TCRs, can be extremelyuseful in immunology, e.g., immunotherapy research and development.

The disclosure also provides methods for multiplexed display andscreening of antigens (e.g., as components of ligands) that bind to aTCR target. In some embodiments, the antigens are displayed on a cellsurface using any of the cell display methods described herein. In someembodiments the antigens are complexed in an MHC complex and displayedon the cell population surfaces.

Antigens that specifically bind to T-cell receptors (TCRs) can beidentified using various detection methods, including isolation of thecells and sequencing of the introduced antigen sequences oridentification by hybridization, e.g., on an array. In other aspects,the barcodes associated with a specific displayed antigen may beidentified and used to identify the antigens that selectively bind to aTCR. The barcodes may be identified, e.g., using sequencing and/or arrayhybridization.

In some embodiments, the cells that encode engineered peptide antigensthat selectively bind to one or more targets of interest from the cellsare identified and/or isolated using a barcode associated with thepeptide. In specific embodiments, the barcode is used to further isolateand/or analyze the cells expressing the peptides identified aspotentially elucidating the binding of an antigen to a TCR. In suchembodiments, the barcode can be used as a “handle” to pull out the cellsof interest for further analysis.

In some embodiments, the method comprises producing via genome editing apopulation or library of edited cells each displaying a singleengineered peptide antigen on its surface, wherein the differentengineered peptide antigens are created using nuclease editing and aresubsequently displayed on the surface of different cells. In otherembodiments, the editing method results in a population or library ofedited cells, where each edited cell displays a plurality of differentengineered peptide antigens on its surface. The cells thus can expressone or more engineered peptide antigens that are displayed on the cellsurface of a single cell of the population, optionally in one or moreMHCs (e.g., HLAs)

In some embodiments, the disclosure provides a method for displaying anengineered peptide antigen on a cell surface, the method comprisingediting a cell using a nucleic acid-directed nuclease to create anucleic acid encoding an engineered putative HLA and incubating anedited cell under conditions sufficient for expressing the engineeredHLA.

In some embodiments, the cells of the library display at least 10²engineered peptide antigens. In some embodiments, the cell displays atleast 10³ engineered peptide antigens. In some embodiments, the celldisplays at least 10⁴ engineered peptide antigens. In some embodiments,the cell displays at least 10⁵ engineered peptide antigens, at least10^(δ) engineered peptide antigens or more. In some embodiments, thedisclosure provides a library of any of the cells described herein. Insome embodiments, the library has at least 10⁸ different members. Insome embodiments, the library has at least 2, at least 5, at least 10,at least 50, at least 100, at least 1000, at least 10,000, at least100,000, at least 1,000,000, at least 10⁷, at least 10⁸, at least 10⁹,at least 10¹⁰ or at least 10¹¹ cells.

In some embodiments, the disclosure provides populations or libraries ofedited cells, wherein the cells encode different engineered peptideantigens and variants thereof, and wherein the variants also comprise abinding motif capable of coupling a binding target. In some embodiments,the binding motif is a biotinylation motif. In some embodiments, thelibrary has at least 10⁸ different members. In some embodiments, thelibrary has at least 2, at least 5, at least 10, at least 50, at least100, at least 1000, at least 10,000, at least 100,000, at least1,000,000, at least 10⁷, at least 10⁸, at least 10⁹, at least 10¹⁰ or atleast 10¹¹ members.

Methods of editing that may be used to generate the libraries orpopulations of cells are described in detail below, as are the cellprocessing modules and instruments used to perform the nuclease-directedgenome editing.

The antigens displayed on the edited cells in the libraries can be anylength between 3-50 amino acids and are preferably between 5-20 aminoacids. In specific aspects, the amino acid peptides are displayed in amanner that allows the appropriate presentation of the antigenic regionof a peptide, e.g., 8-11 amino acids that are known to be available inan MHC on the cell surface.

T-Cell Receptors

T-cell receptors (TCRs) are structurally similar to immunoglobulins, areencoded by homologous genes, and are assembled by somatic recombinationfrom sets of gene segments similar to recombination of immunoglobulingenes. TCR loci have roughly the same number of V gene segments but moreJ gene segments, and there is greater diversification of the junctionsbetween gene segments during gene rearrangement. Moreover, functionalTCRs are not known to diversify their V genes after rearrangementthrough somatic hypermutation. This leads to a TCR in which the highestdiversity is in the central part of the receptor, which contacts thebound antigen of the ligand.

TCR α and β chains each consist of a variable (V) amino-terminal regionand a constant (C) region. The organization of the TCRα and TCRβ loci isshown in FIG. 1. The TCRα locus, like those for the immunoglobulin lightchains, contains V and J gene segments (V_(α), and J_(α)). The TCRβlocus, like that for the immunoglobulin heavy-chain, contains D genesegments in addition to V_(β) and J_(β) gene segments.

The TCR gene segments rearrange during T-cell development to formcomplete V-domain exons (FIG. 2). The TCR gene segments are flanked byheptamer and nonamer recombination signal sequences (RSSs) that arehomologous to those flanking immunoglobulin gene

A further shared feature of immunoglobulin and TCR gene rearrangement isthe presence of P- and N-nucleotides in the junctions between the V, D,and J gene segments of the rearranged TCRβ gene. In T cells, P- andN-nucleotides are also added between the V and J gene segments of allrearranged TCRα genes, whereas only about half the V-J joints inimmunoglobulin light-chain genes are modified by N-nucleotide additionand these are often left without any P-nucleotides as well.

The ligand for the TCR is usually a peptide bound to an MHC molecule.Most of the variability of the TCR ligand is thus in the bound antigenicpeptide occupying the center of the surface in contact with thereceptor. In fact, the three-dimensional structure of theantigen-recognition site of a TCR looks much like that of an antibodymolecule.

The structural diversity of TCRs is mainly attributable to combinatorialand junctional diversity generated during the process of generearrangement. The variability in TCR chains is focused on thejunctional region encoded by V, D, and J gene segments and modified byP- and N-nucleotides. The TCRα locus contains many more J gene segmentsthan either of the immunoglobulin light-chain loci: in humans, 61 J_(α)gene segments are distributed over about 80 kb of DNA, whereasimmunoglobulin light-chain loci have only five J gene segments at most.Because the TCRα locus has so many J gene segments, the variabilitygenerated in this region is even greater for TCRs than forimmunoglobulins. This region encodes the CDR3 loops in immunoglobulinsand TCRs that form the center of the antigen-binding site. Thus, thecenter of the TCR will be highly variable, whereas the periphery will besubject to relatively little variation.

A minority of T cells bear TCRs composed of γ and δ chains. The clusterof gene segments encoding the δ chain is found entirely within the TCRαlocus, between the V_(α) and the J_(α) gene segments. See FIG. 3.Because all V_(α) gene segments are oriented such that rearrangementwill delete the intervening DNA, any rearrangement at the α locusresults in the loss of the δ locus. There are substantially fewer V genesegments at the TCRγ and TCRδ loci than at either the TCRα or TCRβ locior at any of the immunoglobulin loci. Increased junctional variabilityin the δ chains may compensate for the small number of V gene segmentsand has the effect of focusing almost all of the variability in the γ:δreceptor in the junctional region. As we have seen, the amino acidsencoded by the junctional regions lie at the center of the TCR bindingsite. In humans, the TCRγ and TCRδ loci, like the TCRα and TCRβ loci,have discrete V, D, and J gene segments, and C genes.

T cells bearing γ:δ receptors are a distinct lineage of T cells whosefunctions are at present unknown. The ligands for these receptors arealso largely unknown. Some γ:δ TCRs appear to be able to recognizeantigen directly, much as antibodies do, without the requirement forpresentation by an MHC molecule or processing of the antigen.Accordingly, the co-expression of an MHC molecule with a putativeantigen is optional.

Cell Surface Display

Various display technologies can be used with the cell libraries andpopulations generated by the methods and instrumentation describedherein, including yeast surface display technologies, mammalian cellsurface display technologies, and bacterial surface displaytechnologies. Cell surface display technologies include, but are notlimited to, those disclosed in U.S. Pat. Nos. 8,883,692; 8,685,893; and6,699,658; U.S. Pat. Pub. Nos. 20170218382; 20170088611; 20150307560;20150203834; 20140221621; 20140031292; 20140235476, 20140221621;20130184177; 20110008883; No. 20100233195; 20100210473; 20100216659;20090280560; 20090111126; and 20040146976. Bacterial cells, yeast cellsand mammalian cells can all be used for cell surface display.

In certain embodiments, immobilization of an engineered peptide antigento a cell surface may involve specific interactions between theengineered peptide antigen and a binding motif on the engineered peptideantigen.

The engineered peptide antigens of the invention can be expressed in anycell amenable to editing and surface display, and the invention embracesany prokaryotic or eukaryotic cell, including bacterial cells, yeastcells (e.g., Saccharomyces and/or Picchia species), insect cells,Xenopus cells, and mammalian cells. Cells that are particularly suitedfor expression of the fusion proteins of the invention are E. Coli., S.cerevisiae, CHO and 293T cells. The cells may be ‘wild type’ cells orthe cells may be optimized for a particular characteristic or for aparticular enzyme function that may aid in protein expression. Optimizedor engineered cells include cells that have an optimized capability totake up and maintain nucleic acids, cells that have increased proteinsynthesis capability, and/or cells that have increased protein secretioncapability. Cells that maintain the integrity of the edited nucleic acidand the synthesized proteins are particularly useful.

In specific aspects, the edited cells comprise a binding target on theirsurface, and the cells are incubated under conditions resulting insecretion of the engineered peptide antigen, wherein the engineeredpeptide antigen binds to a binding target, thereby displaying theengineered peptide antigen on the cell surface.

A commonly used organism for protein display is yeast. Yeast displayoffers the advantage over bacteria-based technologies in that yeast canprocess proteins that require endoplasmic reticulum (ER)-specificpost-translational processing for efficient folding and activity. Whilemammalian cell display also facilitates post-translational processing,yeast offers the advantage of ease of generation of nucleic acidlibraries as the vectors can be simpler, and yeast allow for an easierintroduction of editing machinery (e.g., editing vectors) into thecells. Most yeast expression fusion proteins are based on GPI(Glycosyl-Phosphatidyl-Inositol) anchor proteins which play importantroles in the surface expression of cell-surface proteins and areessential for the viability of the yeast. One such anchorprotein—alpha-agglutinin—consists of a core subunit encoded by AGA1 andis linked through disulfide bridges to a small binding subunit encodedby AGA2. Proteins encoded by the nucleic acid libraries described hereincan be introduced on the N-terminal region of AGA1 or on the C terminalor N-terminal region of AGA2. These fusion patterns will result in thedisplay of the polypeptide on the yeast cell surface.

In some embodiments, fusion proteins for yeast display include anengineered peptide antigen fused to the N-terminal or C-terminal part ofa protein capable of anchoring in a eukaryotic cell wall (e.g.,a-agglutinin, AGA1, Flo1 or major cell wall protein of lower eukaryotes;see U.S. Pat. Nos. 6,027,910 and 6,114,147 which are hereby incorporatedby reference), for example, proteins fused with the GPI fragment of Flo1or to the Flo1 functional domain (Kondo et al., Appl. MicroBiol.Biotech., 64: 28-40 (2004)).

In addition to surface display methods based on established fusionproteins comprising a GPI anchor motif, the invention also embracesdisplay methods based on novel fusion proteins comprising a modified GPIanchor motif. Fusion proteins of the invention may comprise a protein tobe displayed (e.g., one or more engineered peptide antigens, bindingtargets, molecular targets, substrates, etc., or any combinationthereof), a GPI anchor and appropriate signaling sequences, which may bepost-translationally modified when the fusion protein is expressed inyeast. As a protein containing the GPI anchor and C-terminal signalingsequence is trafficked through the ER, a hydrophobic region on theC-terminal signal sequence adjacent to the GPI anchor becomes embeddedin the ER membrane, where it is cleaved by an ER protease. As the ERprotease cleaves this C-terminal signal sequence, it simultaneouslyattaches a preformed GPI anchor to the new C-terminus of the engineeredpeptide antigen (e.g., binding target, molecular target, substrate,etc., or any combination thereof) ultimately resulting in the display ofthe protein (e.g., binding target, molecular target, substrate, etc., orany combination thereof) on the cell surface (See, e.g., Kondo et al.,cited above). The invention embraces C-terminal sequences with improvedprocessing properties resulting in the improved display of fusionproteins comprising the GPI-anchor proteins. Improved display comprisesan increase in the number of displayed proteins and/or an increase inthe number of correctly expressed proteins. In some embodiments,C-terminal sequences with improved processing properties are evolved byscreening libraries containing variant C-terminal sequences according totechniques known in the art.

In some embodiments, the disclosure provides a method for displaying anengineered peptide antigen on a cell, the method comprising incubatingan edited cell comprising a first nucleic acid under conditionssufficient for expressing an engineered peptide antigen encoded by thefirst nucleic acid, wherein the cell displays a first binding target,wherein the engineered peptide antigen comprises a binding motif and asecond binding target is coupled to the binding motif when theengineered peptide antigen is expressed, and, wherein the expressedengineered peptide antigen is secreted from the cell and displayed onthe cell surface via binding of the second binding target to the firstbinding target. In some embodiments, the first binding target is anavidin-like protein. In some embodiments, the second binding target isbiotin. In some embodiments the binding motif is a biotinylationpeptide. In some embodiments, coupling of the second binding target isdone by a coupling enzyme. In some embodiments, the coupling enzyme is abiotin ligase.

In some embodiments, the disclosure provides a method for generating alibrary of edited cells comprising engineered (edited) peptide antigensdisplayed on the cell surfaces of the cells, the method comprisingintroducing a plurality of editing vectors into a population of cells,creating conditions to allow the editing vectors to edit nucleic acidsin the cells; and creating conditions where the edited cells express theengineered peptide antigens and display the engineered peptide antigenson the cell surfaces, wherein the vectors comprise a nuclease, and adonor nucleic acid sequence comprising an edit in the coding region ofthe antigen to be engineered. In specific aspects, the encodedengineered peptide antigens comprise a unique polypeptide linked to animmobilization peptide, wherein the immobilization peptide comprises afirst binding motif that selectively binds to a second binding motifpresent on the cell surface of the edited cells, and the engineeredpeptide antigens are expressed under conditions sufficient for bindingof the first binding motif to the second binding motif on the cellsurface. The immobilization peptide may also or alternatively comprise,for example, a transmembrane polypeptide, a polypeptide membrane anchor,a GPI-linked polypeptide or a natural surface polypeptide.

In some embodiments, the disclosure provides a method for generating alibrary of edited cells expressing engineered peptide antigens displayedon a cell surface, the method comprising introducing a plurality ofvectors into a population of cells, wherein the vectors comprise anucleic acid-guided nuclease, a guide RNA, and a donor nucleic acidcomprising an edit in the coding region of the protein to be engineered.In specific aspects, the antigens to be edited are encoded engineeredpeptide antigens that comprise a unique polypeptide linked to animmobilization peptide, wherein the immobilization peptide comprises afirst binding motif that selectively binds to a second binding motifpresent on the cell surface of the edited cells, and the engineeredpeptide antigens are expressed under conditions sufficient for bindingof the first binding motif to the second binding motif on the cellsurface.

In the aspects that comprise the use of an immobilization peptide orother moiety comprising a binding motif, the peptide or motif can belinked to the C-terminus or the N-terminus of the engineered peptideantigen.

In some embodiments, the engineered peptide antigen further comprises aleader peptide. The leader peptide or secretion peptide may beproteolytically removed from the mature protein concomitant orimmediately following export of the protein into the lumen ofintracellular compartment along the secretory pathway. The leaderpeptide may be a naturally occurring sequence or a synthetic sequence.

The edited cell library can have at least 2, at least 5, at least 10, atleast 50, at least 100, at least 1000, at least 10,000, at least100,000, at least 1,000,000, at least at least 10⁷, at least 10⁸, atleast 10⁹, at least 10¹⁰ or at least 10¹¹ cells comprising one or moreengineered peptide antigens.

In some embodiments the expression of the engineered peptide antigens inthe cells is inducible or transient. In some embodiments, no inductionstep is necessary and incubating the cell results in the expression ofthe engineered peptide antigen. In some embodiments, engineered peptideantigens comprising a first binding motif are secreted and bind to asecond binding motif present on the cell surface, thereby displaying theengineered peptide antigen on the cell surface. In some embodiments, thefirst binding motif is avidin, streptavidin or neutravidin and thesecond binding motif is biotin. In some embodiments, avidin iscovalently conjugated to the cell surface (e.g., directly orindirectly). Yet in some embodiments, the first binding target isexpressed by the cell and displayed at the cell surface. For example,one of the binding targets may be expressed by the cell as a fusionprotein such as a cell wall or a membrane fusion protein and displayedat the surface of the cell.

Screening Methods

The methods of the disclosure may be useful to identify one or morepeptides that selectively bind to a TCR. By providing a system thatcreates a cell library with engineered peptide antigens displayed on thesurface of the cells in which they are expressed, cells that expressengineered peptide antigens can be identified using any assay that canbe performed on a cell surface (e.g., performed on a cellularpreparation to detect one or more molecules that are displayed on thecell surface). The methods of the disclosure can be used to screenlibraries expressing engineered peptide antigen variants to identify oneor more TCRs that selectively bind to the antigen(s).

An embodiment of the disclosure provides a method for selecting cellsdisplaying engineered peptide antigens with desirable affinity orspecificity for a target TCR, e.g., a known TCR or an orphan TCR. Someaspects of the invention relate to methods to screen for cellsexpressing an antigen that can interact with a specific target molecule(e.g., a known TCR or orphan TCR) with a desired specificity.

In some embodiments, the disclosure provides an antigen screening methodcomprising expressing an engineered peptide antigen in a cell editedusing a nuclease, wherein the expressed engineered peptide antigen issecreted and displayed on the cell surface as a component of a ligandspecific for a TCR and evaluating the binding of the ligand to one ormore TCRs. Upon identification of a particular TCR and/or peptide, thesequences can be sequenced, e.g., using next-generation sequencing suchas Illumina HiSeq or MiSeq. In other aspects, the specific TCR and/orpeptide can be identified through the detection of a barcode that isassociated with a particular TCR and/or peptide.

In some embodiments, the disclosure provides an antigen screening methodcomprising expressing an engineered peptide antigen in a cell editedusing a nucleic-acid directed nuclease (e.g., an RNA-directed nucleasesuch as a CRISPR nuclease). The expressed engineered peptide antigensare secreted and displayed on the cell surface as a component of aligand specific for a TCR and evaluating the binding of the ligand tothe one or more TCRs.

Expression of Edited Proteins

The engineered peptide antigens in the edited cells of the invention canbe expressed from the edited nucleic acids using methods known in theart. In some embodiments, protein expression is constitutive.Constitutive expression covers both expression from nucleic acids thathave been integrated into the genome and expression from nucleic acidsthat are located on episomal vectors. In some embodiments, expression isinitiated by an inducible event. In some embodiments, edited nucleicacids that encode the engineered peptide antigens are operably connectedto an initiator sequence that regulates expression of the engineeredpeptide antigen. Initiator sequences that can induce expression areknown in the art and include inducible promoters. In some embodimentsprotein expression is induced. In some embodiments, protein expressionoccurs when the cell comprising a nucleic acid encoding the protein isincubated and no separate induction step is required.

Cell Libraries

Libraries of the invention include libraries of edited cells expressingunique engineered peptide antigens. The cells of the libraries arepreferably edited using a nuclease, and more preferably using one ormore nucleases (e.g., a nucleic acid-directed nuclease) in an automatedmulti-module cell editing instrument as described in more detail herein.

In some embodiments, the library provides edited cells with a highdensity of engineered peptide antigens immobilized on the cell surface.In some embodiments, the high density is accomplished by bindingmultiple engineered polypeptides expressed in a cell to a cell-surfacebinding target. In some embodiments, the number of engineered peptideantigens that are displayed per cell is greater than 10³, greater than10⁴, greater than 10⁵, greater than 10⁶, greater than 10⁷, or greaterthan 10⁸ engineered peptide antigens per cell. In some embodiments, theimmobilization peptide is a biotinylation peptide. The antigensdisplayed may be a single peptide antigen or two or more peptideantigens depending on the display strategy for the cells. In someembodiments, the immobilization peptide is a transmembrane protein. Insome embodiments, the immobilization peptide comprises a GPI anchor. Insome embodiments, the immobilization peptide is a peptide that isnaturally present on the cell surface. In some embodiments, theimmobilization peptide is a peptide that binds one or more moleculesnaturally present on the cell surface (e.g., surface carbohydrates orproteins on the cell surface).

In some embodiments, libraries of binding proteins may be evaluated orscreened to identify and/or isolate variants that bind to one or moreTCR targets. Methods of the invention may be designed to identifyengineered peptide antigens that have affinities for a particular TCRgreater than a binding affinity represented by a dissociation constantof about 10⁻⁷ M, about 10⁻⁸ M, about 10⁻⁹ M, about 10⁻¹⁰ M, about 10⁻¹¹M, about 10⁻¹² M, about 10⁻¹³ M, about 10⁻¹⁴ M or about 10⁻¹⁵ M. In someembodiments, methods of the invention may be designed to identify targetpeptide sequences that have affinities for a TCR greater than a bindingaffinity represented by a dissociation constant of about 10⁻⁷ M, about10⁻⁸ M, about 10⁻⁹ M, about 10⁻¹⁰ M, about 10⁻¹¹ M, about 10⁻¹² M, about10⁻¹³ M, about 10⁻¹⁴ M or about 10⁻¹⁵M.

Nuclease-Directed Genome Editing

In embodiments, the automated instrument described herein utilizes anuclease-directed genome editing system for introducing edits to apopulation of cells allowing the engineering of proteins for cellsurface display. Multiple different nuclease-based systems exist forproviding edits into an organism's genome, and each can be used ineither single editing systems, sequential editing systems (e.g., usingdifferent nuclease-directed systems sequentially to provide two or moregenome edits in a cell) and/or recursive editing systems, (e.g.,utilizing a single nuclease-directed system to introduce two or moregenome edits in a cell). Exemplary nuclease-directed genome editingsystems are described herein, although a person of skill in the artwould recognize upon reading the present disclosure that other suchediting instruments are also useful in the creation of populations ofcells for cell surface display of engineered peptide antigens.

It should be noted that the automated editing instruments as set forthherein can use the nucleases for cleaving the genome, introduction of anedit into a target region, or both.

In particular aspects of the invention, the nuclease editing system isan inducible system that allows control of the timing of the editing.The ability to modulate nuclease activity can reduce off-target cleavageand facilitate precise genome engineering. Numerous different induciblesystems can be used with the instrument and systems of the disclosure,as will be apparent to one skilled in the art upon reading the presentdisclosure.

In certain aspects, cleavage by a nuclease can be used with theinstruments and systems of the invention to select cells with a genomicedit at a target region. For example, cells that have been subjected toa genomic edit that removes a particular nuclease recognition site(e.g., via homologous recombination) can be selected using theinstruments described herein by exposing the cells to the nucleasefollowing the edit. The DNA in the cells without the genome edit will becleaved and subsequently will have limited growth and/or perish, whereasthe cells that received the genome edit removing the nucleaserecognition site will not be affected by the subsequent exposure to thenuclease.

In other aspects, cells for editing may be treated in some fashion tocleave the genome prior to introduction of the cells to the instrument,and the instrument used for automated introduction of desired genomeedits in such cells. The initial cleavage can be performed by the sameor a different enzyme than the one used for the initial cleavage event.

When the cell or population of cells comprising nucleic acid-guidednuclease encoding DNA is in the presence of the inducer molecule,expression of the nuclease can occur. For example, CRISPR-nucleaseexpression can be repressed in the presence of a repressor molecule.When the cell or population of cells comprising nucleic acid-guidednuclease encoding DNA is in the absence of a molecule that repressesexpression of the CRISPR-nuclease, expression of the CRISPR-nuclease canoccur.

For example, inducible systems for editing using RNA-guided nucleasehave been described, which use chemical induction to limit the temporalexposure of the cells to the RNA-guided nuclease. Dow, et al., NatureBiotechnology, 33:390-394 (2015); see also inducible lentiviralexpression vectors available at Dharmacon, GE Life Sciences, Lafayette,Colo., For additional techniques, see e.g., Campbell, Biochem J.,473(17): 2573-89 (2010).

In other examples, a virus-inducible nuclease can be used to induce geneediting in cells. See, e.g., Don, Antiviral Res., 130:50-57 (2016). Inanother example, for inducible expression of nucleic acid directednucleases, variants can be switched on and off in human cells with4-hydroxytamoxifen (4-HT) by fusing the nuclease with thehormone-binding domain of the estrogen receptor (ERT2), Liu, et al.,Nature Chemical Biology, 12:980-87 (2016).

Zinc-finger nucleases (ZFNs) are artificial restriction enzymesgenerated by fusing a zinc finger DNA-binding domain to a DNA-cleavagedomain. Zinc finger domains can be engineered to target specific targetregions in an organism's genome. See, e.g., Urnov, et al., NatureReviews Genetics 11, 636-646 (2010). Using the endogenous DNA repairmachinery of an organism, ZFNs can be used to precisely alter a targetregion of the genome. ZFNs can be used to disable dominant mutations inheterozygous individuals by producing double-strand breaks (“DSBs”) inthe DNA in the mutant allele, which will, in the absence of a homologoustemplate, be repaired by non-homologous end-joining (NHEJ). NHEJ repairsDSBs by joining the two ends together and usually produces no mutations,provided that the cut is clean and uncomplicated. Dural, et al., NucleicAcids Res. 33 (18): 5978-90 (2005). This repair mechanism can be used toinduce errors in the genome via indels or chromosomal rearrangement,often rendering the gene products coded at that location non-functional.

Alternatively, DNA can be introduced into a genome in the presence ofexogenous double-stranded DNA fragments using homology dependent repair(HDR). The dependency of HDR on a homologous sequence to repair DSBs canbe exploited by inserting a desired sequence within a sequence that ishomologous to the flanking sequences of a DSB which, when used as atemplate by HDR system, would lead to the creation of the desired changewithin the genomic region of interest.

Multiple pairs of ZFNs can also be used to completely remove entirelarge segments of genomic sequence (Lee. et al., Genome Res. 20(1):81-89 (2009). Expanded CAG/CTG repeat tracts are the genetic basis formore than a dozen inherited neurological disorders includingHuntington's disease, myotonic dystrophy, and several spinocerebellarataxias. It has been demonstrated in human cells that ZFNs can directDSBs to CAG repeats and shrink the repeat from long pathological lengthsto short, less toxic lengths (Mittelman, et al., PNAS USA, 106(24):9607-12 (2009)).

Meganucleases were identified in the 1990s, and subsequent work hasshown that they are particularly promising tools for genome editing, asthey are able to efficiently induce homologous recombination, generatemutations in coding or non-coding regions of the genome, and alterreading frames of the coding regions of genomes. See, e.g., Epinat, etal., Nucleic Acids Research, 31(11):2952-62 (2003). The high specificityof meganucleases gives them a high degree of precision and much lowercell toxicity than other naturally occurring restriction enzymes.

Transcription activator-like effector nucleases (TALENs) are restrictionenzymes that can be engineered to cut specific sequences of DNA. Theyare made by fusing a TAL effector DNA-binding domain to a DNA cleavagedomain (a nuclease which cuts DNA strands). Transcription activator-likeeffectors (TALEs) can be engineered to bind to practically any desiredDNA sequence, so when combined with a nuclease, DNA can be cut atspecific locations. (See, e.g., Miller, et al., Nature Biotechnology,29(2): 143-48 (2011); Boch, Nature Biotechnology, 29(2): 135-36 (2011)).

Like ZFNs, TALEN can edit genomes by inducing DSBs. The TALEN-createdsite-specific DSBs at target regions are repaired through NHEJ or HDR,resulting in targeted genome edits. TALENs can be used to introduceindels, rearrangements, or to introduce DNA into a genome through NHEJin the presence of exogenous double-stranded DNA fragments.

A recent discovery for editing live cells involves nucleic acid-guidednuclease (e.g., RNA-guided nuclease) editing. A nucleic acid-guidednuclease complexed with an appropriate synthetic guide nucleic acid in acell can cut the genome of the cell at a desired location. The guidenucleic acid helps the nucleic acid-guided nuclease recognize and cutthe DNA at a specific target sequence. By manipulating the nucleotidesequence of the guide nucleic acid, the nucleic acid-guided nuclease maybe programmed to target any DNA sequence for cleavage as long as anappropriate protospacer adjacent motif (PAM) is nearby. In certainaspects, the nucleic acid-guided nuclease editing system may use twoseparate guide nucleic acid molecules that combine to function as aguide nucleic acid, e.g., a CRISPR RNA (crRNA) and trans-activatingCRISPR RNA (tracrRNA). In other aspects, the guide nucleic acid may be asingle guide nucleic acid that includes both the crRNA and tracrRNAsequences.

In general, a guide nucleic acid (e.g., gRNA) complexes with acompatible nucleic acid-guided nuclease and can then hybridize with atarget sequence, thereby directing the nuclease to the target sequence.A guide nucleic acid can be DNA or RNA; alternatively, a guide nucleicacid may comprise both DNA and RNA. In some embodiments, a guide nucleicacid may comprise modified or non-naturally occurring nucleotides. Incases where the guide nucleic acid comprises RNA, the gRNA may beencoded by a DNA sequence on a polynucleotide molecule such as aplasmid, linear construct, or the coding sequence may reside within anediting cassette and is under the control of a constitutive promoter,or, in some embodiments and preferably, an inducible promoter asdescribed below.

A guide nucleic acid comprises a guide sequence, where the guidesequence is a polynucleotide sequence having sufficient complementaritywith a target sequence to hybridize with the target sequence and directsequence-specific binding of a complexed nucleic acid-guided nuclease tothe target sequence. The degree of complementarity between a guidesequence and the corresponding target sequence, when optimally alignedusing a suitable alignment algorithm, is about or more than about 50%,60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment maybe determined with the use of any suitable algorithm for aligningsequences. In some embodiments, a guide sequence is about or more thanabout 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.In some embodiments, a guide sequence is less than about 75, 50, 45, 40,35, 30, 25, 20 nucleotides in length. Preferably the guide sequence is10-30 or 15-20 nucleotides long, or 15, 16, 17, 18, 19, or 20nucleotides in length.

In the present methods and compositions, the guide nucleic acid isprovided as a sequence to be expressed from a plasmid or vector andcomprises both the guide sequence and the scaffold sequence as a singletranscript under the control of a promoter, and in some embodiments, aninducible promoter. The guide nucleic acid can be engineered to target adesired target sequence by altering the guide sequence so that the guidesequence is complementary to a desired target sequence, thereby allowinghybridization between the guide sequence and the target sequence. Ingeneral, to generate an edit in the target sequence, the gRNA/nucleasecomplex binds to a target sequence as determined by the guide RNA, andthe nuclease recognizes a proto spacer adjacent motif (PAM) sequenceadjacent to the target sequence. The target sequence can be anypolynucleotide endogenous or exogenous to a prokaryotic or eukaryoticcell, or in vitro. For example, the target sequence can be apolynucleotide residing in the nucleus of a eukaryotic cell. A targetsequence can be a sequence encoding a gene product (e.g., a protein) ora non-coding sequence (e.g., a regulatory polynucleotide, an intron, aPAM, or “junk” DNA).

The guide nucleic acid may be part of an editing cassette that encodesthe donor nucleic acid. Alternatively, the guide nucleic acid may not bepart of the editing cassette and instead may be encoded on the engine orediting vector backbone. For example, a sequence coding for a guidenucleic acid can be assembled or inserted into a vector backbone first,followed by insertion of the donor nucleic acid in, e.g., the editingcassette. In other cases, the donor nucleic acid in, e.g., an editingcassette can be inserted or assembled into a vector backbone first,followed by insertion of the sequence coding for the guide nucleic acid.In yet other cases, the sequence encoding the guide nucleic acid and thedonor nucleic acid (inserted, for example, in an editing cassette) aresimultaneously but separately inserted or assembled into a vector. Inyet other embodiments, the sequence encoding the guide nucleic acid andthe sequence encoding the donor nucleic acid are both included in theediting cassette.

The target sequence is associated with a protos-spacer mutation (PAM),which is a short nucleotide sequence recognized by the gRNA/nucleasecomplex. The precise preferred PAM sequence and length requirements fordifferent nucleic acid-guided nucleases vary; however, PAMs typicallyare 2-7 base-pair sequences adjacent or in proximity to the targetsequence and, depending on the nuclease, can be 5′ or 3′ to the targetsequence. Engineering of the PAM-interacting domain of a nucleicacid-guided nuclease may allow for alteration of PAM specificity,improve target site recognition fidelity, decrease target siterecognition fidelity, or increase the versatility of a nucleicacid-guided nuclease. In certain embodiments, the genome editing of atarget sequence both introduces a desired DNA change to a targetsequence, e.g., the genomic DNA of a cell, and removes, mutates, orrenders inactive a proto-spacer mutation (PAM) region in the targetsequence. Rendering the PAM at the target sequence inactive precludesadditional editing of the cell genome at that target sequence, e.g.,upon subsequent exposure to a nucleic acid-guided nuclease complexedwith a synthetic guide nucleic acid in later rounds of editing. Thus,cells having the desired target sequence edit and an altered PAM can beselected using a nucleic acid-guided nuclease complexed with a syntheticguide nucleic acid complementary to the target sequence. Cells that didnot undergo the first editing event will be cut rendering adouble-stranded DNA break, and thus will not continue to be viable. Thecells containing the desired target sequence edit and PAM alterationwill not be cut, as these edited cells no longer contain the necessaryPAM site and will continue to grow and propagate.

The range of target sequences that nucleic acid-guided nucleases canrecognize is constrained by the need for a specific PAM to be locatednear the desired target sequence. As a result, it often can be difficultto target edits with the precision that is necessary for genome editing.It has been found that nucleases can recognize some PAMs very well(e.g., canonical PAMs), and other PAMs less well or poorly (e.g.,non-canonical PAMs). Because the methods disclosed herein allow foridentification of edited cells in a background of unedited cells, themethods allow for identification of edited cells where the PAM is lessthan optimal; that is, the methods for identifying edited cells hereinallow for identification of edited cells even if editing efficiency isvery low. Additionally, the present methods expand the scope of targetsequences that may be edited since edits are more readily identified,including cells where the genome edits are associated with lessfunctional PAMs.

As for the nuclease component of the nucleic acid-guided nucleaseediting system, a polynucleotide sequence encoding the nucleicacid-guided nuclease can be codon optimized for expression in particularcell types, such as archaeal, prokaryotic or eukaryotic cells.Eukaryotic cells can be yeast, fungi, algae, plant, animal, or humancells. Eukaryotic cells may be those of or derived from a particularorganism, such as a mammal, including but not limited to human, mouse,rat, rabbit, dog, or non-human mammals including non-human primates. Thechoice of nucleic acid-guided nuclease to be employed depends on manyfactors, such as what type of edit is to be made in the target sequenceand whether an appropriate PAM is located close to the desired targetsequence. Nucleases of use in the methods described herein include butare not limited to Cas 9, Cas 12/CpfI, MAD2, or MAD7 or other MADzymes.As with the guide nucleic acid, the nuclease may be encoded by a DNAsequence on a vector (e.g., the engine vector) and be under the controlof a constitutive or inducible promoter. In some embodiments, thesequence encoding the nuclease is under the control of an induciblepromoter, and the inducible promoter may be separate from but the sameas the inducible promoter controlling transcription of the guide nucleicacid; that is, a separate inducible promoter drives the transcription ofthe nuclease and guide nucleic acid sequences but the two induciblepromoters may be the same type of inducible promoter (e.g., both are pLpromoters). Alternatively, the inducible promoter controlling expressionof the nuclease may be different from the inducible promoter controllingtranscription of the guide nucleic acid; that is, e.g., the nuclease maybe under the control of the pBAD inducible promoter, and the guidenucleic acid may be under the control of the pL inducible promoter.

Another component of the nucleic acid-guided nuclease system is thedonor nucleic acid. In some embodiments, the donor nucleic acid is onthe same polynucleotide (e.g., editing vector or editing cassette) asthe guide nucleic acid and may be (but not necessarily) under thecontrol of the same promoter as the guide nucleic acid (e.g., a singlepromoter driving the transcription of both the guide nucleic acid andthe donor nucleic acid). The donor nucleic acid is designed to serve asa template for homologous recombination with a target sequence nicked orcleaved by the nucleic acid-guided nuclease as a part of thegRNA/nuclease complex. A donor nucleic acid polynucleotide may be of anysuitable length, such as about or more than about 20, 25, 50, 75, 100,150, 200, 500, or 1000 nucleotides in length. In certain preferredaspects, the donor nucleic acid can be provided as an oligonucleotide ofbetween 20-300 nucleotides, more preferably between 50-250 nucleotides.The donor nucleic acid comprises a region that is complementary to aportion of the target sequence (e.g., a homology arm). When optimallyaligned, the donor nucleic acid overlaps with (is complementary to) thetarget sequence by, e.g., about 20, 25, 30, 35, 40, 50, 60, 70, 80, 90or more nucleotides. In many embodiments, the donor nucleic acidcomprises two homology arms (regions complementary to the targetsequence) flanking the mutation or difference between the donor nucleicacid and the target template. The donor nucleic acid comprises at leastone mutation or alteration compared to the target sequence, such as aninsertion, deletion, modification, or any combination thereof comparedto the target sequence.

Often the donor nucleic acid is provided as an editing cassette, whichis inserted into a vector backbone where the vector backbone maycomprise a promoter driving transcription of the gRNA and the codingsequence of the gRNA, or the vector backbone may comprise a promoterdriving the transcription of the gRNA but not the gRNA itself. Moreover,there may be more than one, e.g., two, three, four, or more guidenucleic acid/donor nucleic acid cassettes inserted into an enginevector, where each guide nucleic acid is under the control of separatedifferent promoters, separate like promoters, or where all guide nucleicacid/donor nucleic acid pairs are under the control of a singlepromoter. In some embodiments the promoter driving transcription of thegRNA and the donor nucleic acid (or driving more than one gRNA/donornucleic acid pair) is an inducible promoter and the promoter drivingtranscription of the nuclease is an inducible promoter as well. Foradditional information regarding editing cassettes, see U.S. Pat. No.9,982,278, and U.S. Ser. Nos. 15/948,789; 15/116,616; 15/948,785;16/056,310; 16/275,439; and 16/275,465.

Inducible editing is advantageous in that singulated cells can be grownfor several to many cell doublings before editing is initiated, whichincreases the likelihood that cells with edits will survive, as thedouble-strand cuts caused by active editing are largely toxic to thecells. This toxicity results both in cell death in the edited colonies,as well as possibly a lag in growth for the edited cells that do survivebut must repair and recover following editing. However, once the editedcells have a chance to recover, the size of the colonies of the editedcells will eventually catch up to the size of the colonies of uneditedcells. Further, a guide nucleic acid may be efficacious directing theedit of more than one donor nucleic acid in an editing cassette; e.g.,if the desired edits are close to one another in a target sequence.

In addition to the donor nucleic acid, an editing cassette may compriseone or more primer sites. The primer sites can be used to amplify theediting cassette by using oligonucleotide primers; for example, if theprimer sites flank one or more of the other components of the editingcassette.

Also, as described above, the donor nucleic acid may comprise—inaddition to the at least one mutation relative to a target sequence—oneor more PAM sequence alterations that mutate, delete or render inactivethe PAM site in the target sequence. The PAM sequence alteration in thetarget sequence renders the PAM site “immune” to the nucleic acid-guidednuclease and protects the target sequence from further editing insubsequent rounds of editing if the same nuclease is used.

In addition, the editing cassette may comprise a barcode. A barcode is aunique DNA sequence that corresponds to the donor DNA sequence such thatthe barcode can identify the edit made to the corresponding targetsequence. The barcode typically comprises four or more nucleotides. Insome embodiments, the editing cassettes comprise a collection of donornucleic acids representing, e.g., gene-wide or genome-wide libraries ofdonor nucleic acids. The library of editing cassettes is cloned intovector backbones where, e.g., each different donor nucleic acid isassociated with a different barcode.

Additionally, in some embodiments, an expression vector or cassetteencoding components of the nucleic acid-guided nuclease system furtherencodes a nucleic acid-guided nuclease comprising one or more nuclearlocalization sequences (NLSs), such as about or more than about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the engineerednuclease comprises NLSs at or near the amino-terminus, NLSs at or nearthe carboxy-terminus, or a combination.

The engine and editing vectors comprise control sequences operablylinked to the component sequences to be transcribed. As stated above,the promoters driving transcription of one or more components of thenucleic acid-guided nuclease editing system may be inducible. A numberof gene regulation control systems have been developed for thecontrolled expression of genes in plant, microbe, and animal cells,including mammalian cells, including the pL promoter (induced by heatinactivation of the CI857 repressor), the pBAD promoter (induced by theaddition of arabinose to the cell growth medium), and the rhamnoseinducible promoter (induced by the addition of rhamnose to the cellgrowth medium). Other systems include the tetracycline-controlledtranscriptional activation system (Tet-On/Tet-Off, Clontech, Inc. (PaloAlto, Calif.); Bujard and Gossen, PNAS, 89(12):5547-5551 (1992)), theLac Switch Inducible system (Wyborski et al., Environ Mol Mutagen,28(4):447-58 (1996); DuCoeur et al., Strategies 5(3):70-72 (1992); U.S.Pat. No. 4,833,080), the ecdysone-inducible gene expression system (Noet al., PNAS, 93(8):3346-3351 (1996)), the cumate gene-switch system(Mullick et al., BMC Biotechnology, 6:43 (2006)), and thetamoxifen-inducible gene expression (Zhang et al., Nucleic AcidsResearch, 24:543-548 (1996)) as well as others. In the present methodsused in the modules and instruments described herein, it is preferredthat at least one of the nucleic acid-guided nuclease editing components(e.g., the nuclease and/or the gRNA) is under the control of a promoterthat is activated by a rise in temperature, as such a promoter allowsfor the promoter to be activated by an increase in temperature, andde-activated by a decrease in temperature, thereby “turning off” theediting process. Thus, in the scenario of a promoter that isde-activated by a decrease in temperature, editing in the cell can beturned off without having to change media; to remove, e.g., an induciblebiochemical in the medium that is used to induce editing.

Automated Cell Editing Instruments and Modules to Create Cell SurfaceDisplay Libraries

Automated Cell Editing Instruments

FIG. 4A depicts an exemplary automated multi-module cell processinginstrument 400 to, e.g., perform one of the exemplary workflowsdescribed above, as well as additional exemplary modules. The instrument400, for example, may be and preferably is designed as a desktopinstrument for use within a laboratory environment. The instrument 400may incorporate a mixture of reusable and disposable elements forperforming various staged processes in conducting automated genomecleavage and/or editing in cells. Illustrated is a gantry 402, providingan automated mechanical motion system (actuator) (not shown) thatsupplies XYZ axis motion control to, e.g., an automated liquid handlingsystem 458 including, e.g., an air displacement pipette as well asmodules of the automated multi-module cell processing instrument 400. Insome automated multi-module cell processing instruments, the airdisplacement pipettor 432 is moved by gantry 402 and the various modulesand reagent cartridges remain stationary; however, in other embodiments,the liquid handling system may stay stationary while the various modulesare moved. Also included in the automated multi-module cell processinginstrument 400 is reagent cartridge 410 comprising reservoirs 412 andtransformation module 430, as well as a wash cartridge 404 comprisingreservoirs 406. The wash cartridge 404 may be configured to accommodatelarge tubes, for example, wash solutions, or solutions that are usedoften throughout an iterative process. In one example, wash cartridge404 may be configured to remain in place when two or more reagentcartridges 410 are sequentially used and replaced. Although reagentcartridge 410 and wash cartridge 404 are shown in FIG. 4A as separatecartridges, the contents of wash cartridge 404 may be incorporated intoreagent cartridge 410. Note in this embodiment transformation module 430is contained within reagent cartridge 410; however, in alternativeembodiments transformation module 430 is contained within its own moduleor may be part of another module, such as a growth module.

The wash and reagent cartridges 404 and 410 in some implementations, aredisposable kits provided for use in the automated multi-module cellediting instrument 400. For example, a user may open and position eachof the reagent cartridge 410 and the wash cartridge 404 within a chassisof the automated multi-module cell editing instrument prior toactivating cell processing.

Also illustrated is the robotic handling system 458 including the gantry402 and air displacement pipettor 432. In some examples, the robotichandling system 458 may include an automated liquid handling system suchas those manufactured by Tecan Group Ltd. of Mannedorf, Switzerland,Hamilton Company of Reno, Nev. (see, e.g., WO2018015544A1), or BeckmanCoulter, Inc. of Fort Collins, Colo. (see, e.g., US20160018427A1).Pipette tips may be provided in a pipette transfer tip supply (notshown) for use with the air displacement pipettor 432.

Components of the cartridges 404, 410, in some implementations, aremarked with machine-readable indicia (not shown), such as bar codes, forrecognition by the robotic handling system 458. For example, the robotichandling system 458 may scan containers within each of the cartridges404, 410 to confirm contents. In other implementations, machine-readableindicia may be marked upon each cartridge 404, 410, and the processingsystem 426 (shown in FIG. 4D) of the automated multi-module cell editinginstrument 400 may identify a stored materials map based upon themachine-readable indicia. The exemplary automated multi-module cellprocessing instrument 400 of FIG. 4A further comprises a cell growthmodule 434. (Note, all modules recited briefly here are described indetail below.) In the embodiment illustrated in FIG. 4A, the cell growthmodule 434 comprises two cell growth vials 418, 420 (described ingreater detail below in relation to FIGS. 5A-5D) as well as a cellconcentration module 422 (described in detail in relation to FIGS.6A-6F). In alternative embodiments, the cell concentration module 422may be separate from cell growth module 434, e.g., in a separate,dedicated module. Also illustrated as part of the automated multi-modulecell processing instrument 400 of FIG. 4A is an optional enrichmentmodule 440, served by, e.g., robotic handing system 458 and airdisplacement pipettor 432. Also seen are an optional nucleic acidassembly/desalting module 414 comprising a reaction chamber or tubereceptacle (not shown) and a magnet 416 to allow for purification ofnucleic acids using, e.g., magnetic solid phase reversibleimmobilization (SPRI) beads (Applied Biological Materials Inc.,Richmond, BC). The cell growth module, cell concentration module,transformation module, enrichment module, reagent cartridge, and nucleicacid assembly module are described in greater detail below.

FIG. 4B is a plan view of the front of the exemplary multi-module cellprocessing instrument 400 depicted in FIG. 4A. Cartridge-based sourcematerials (such as in reagent cartridge 410), for example, may bepositioned in designated areas on a deck 402 of the instrument 400 foraccess by a robotic handling instrument (not shown in this figure). Asillustrated in FIG. 4B, the deck 402 may include a protection sink suchthat contaminants spilling, dripping, or overflowing from any of themodules of the instrument 400 are contained within a lip of theprotection sink. In addition to reagent cartridge 410, also seen in FIG.4B is wash cartridge 404, optional enrichment module 440, and a portionof growth module 434. Also seen in this view is touch screen display450, transformation module controls 438, electronics rack 436, andprocessing system 426.

FIGS. 4C through 4D illustrate multi-module cell processing instruments480 comprising chassis 490 for use in desktop versions the cell editinginstrument 480. For example, the chassis 490 may have a width of about24-48 inches, a height of about 24-48 inches and a depth of about 24-48inches. Chassis 490 may be and preferably is designed to hold multiplemodules and disposable supplies used in automated cell processing.Further, chassis 490 may mount a robotic handling system 458 for movingmaterials between modules.

As illustrated, the chassis 490 includes a cover having a handle 454 andhinges 456 a-456 c for lifting the cover and accessing the interior ofthe chassis 490. A cooling grate 464 allows for air flow via an internalfan (not shown). Further, the chassis 490 is lifted by adjustable feet470 (feet 470 a-c are shown). The feet 470 a-470 c, for example, mayprovide additional air flow beneath the chassis 490. A control button466, in some embodiments, allows for single-button automated startand/or stop of cell processing within the chassis 490.

Inside the chassis 490, in some implementations, a robotic handlingsystem 458 is disposed along a gantry 402 above materials cartridges 404and 410. Control circuitry, liquid handling tubes, air pump controls,valves, thermal units (e.g., heating and cooling units) and othercontrol mechanisms, in some embodiments, are disposed below a deck ofthe chassis 490, in a control box region 468. Also seen in FIG. 4D isenrichment module 440 and nucleic acid assembly module 414 comprising amagnet 416

Although not illustrated, in some embodiments a display screen may bepositioned on the front face of the chassis 490, for example covering aportion of the cover (e.g., see FIG. 4B). The display screen may provideinformation to the user regarding the processing status of the automatedmulti-module cell editing instrument. In another example, the displayscreen may accept inputs from the user for conducting the cellprocessing.

The Rotating Cell Growth Module

FIG. 5A shows one embodiment of a rotating growth vial 500 for use withthe cell growth device described herein. The rotating growth vial is anoptically-transparent container having an open end 504 for receivingliquid media and cells, a central vial region 506 that defines theprimary container for growing cells, a tapered-to-constricted region 518defining at least one light path 510, a closed end 516, and a driveengagement mechanism 512. The rotating growth vial has a centrallongitudinal axis 520 around which the vial rotates, and the light path510 is generally perpendicular to the longitudinal axis of the vial. Thefirst light path 510 is positioned in the lower constricted portion ofthe tapered-to-constricted region 518. Optionally, some embodiments ofthe rotating growth vial 500 have a second light path 508 in the taperedregion of the tapered-to-constricted region 518. Both light paths inthis embodiment are positioned in a region of the rotating growth vialthat is constantly filled with the cell culture (cells+growth media) andis not affected by the rotational speed of the growth vial. The firstlight path 510 is shorter than the second light path 508 allowing forsensitive measurement of OD values when the OD values of the cellculture in the vial are at a high level (e.g., later in the cell growthprocess), whereas the second light path 508 allows for sensitivemeasurement of OD values when the OD values of the cell culture in thevial are at a lower level (e.g., earlier in the cell growth process).Also shown is lip 502, which allows the rotating growth vial to beseated in a growth module (not shown) and further allows for easyhandling for the user.

In some configurations of the rotating growth vial, the rotating growthvial has two or more “paddles” or interior features disposed within therotating growth vial, extending from the inner wall of the rotatinggrowth vial toward the center of the central vial region. In someaspects, the width of the paddles or features varies with the size orvolume of the rotating growth vial, and may range from 1/20 to just over⅓ the diameter of the rotating growth vial, or from 1/15 to ¼ thediameter of the rotating growth vial, or from 1/10 to ⅕ the diameter ofthe rotating growth vial. In some aspects, the length of the paddlesvaries with the size or volume of the rotating growth vial, and mayrange from ⅘ to ¼ the length of the main body of the rotating growthvial, or from ¾ to ⅓ the length of the main body of the rotating growthvial, or from ½ to ⅓ the length of the main body of the rotating growthvial. In other aspects, there may be concentric rows of raised featuresdisposed on the inner surface of the main body of the rotating growthvial arranged horizontally or vertically; and in other aspects, theremay be a spiral configuration of raised features disposed on the innersurface of the main body of the rotating growth vial. In alternativeaspects, the concentric rows of raised features or spiral configurationmay be disposed upon a post or center structure of the rotating growthvial. Though described above as having two paddles, the rotating growthvial may comprise 3, 4, 5, 6 or more paddles, and up to 20 paddles. Thenumber of paddles will depend upon, e.g., the size or volume of therotating growth vial. The paddles may be arranged symmetrically assingle paddles extending from the inner wall of the vial into theinterior of the vial, or the paddles may be symmetrically arranged ingroups of 2, 3, 4 or more paddles in a group (for example, a pair ofpaddles opposite another pair of paddles) extending from the inner wallof the vial into the interior of the vial. In another embodiment, thepaddles may extend from the middle of the rotating growth vial outtoward the wall of the rotating growth vial, from, e.g., a post or othersupport structure in the interior of the rotating growth vial.

The drive engagement mechanism 512 engages with a motor (not shown) torotate the vial. In some embodiments, the motor drives the driveengagement mechanism 512 such that the rotating growth vial is rotatedin one direction only, and in other embodiments, the rotating growthvial is rotated in a first direction for a first amount of time orperiodicity, rotated in a second direction (i.e., the oppositedirection) for a second amount of time or periodicity, and this processmay be repeated so that the rotating growth vial (and the cell culturecontents) are subjected to an oscillating motion. The first amount oftime and the second amount of time may be the same or may be different.The amount of time may be 1, 2, 3, 4, 5, or more seconds, or may be 1,2, 3, 4 or more minutes. In another embodiment, in an early stage ofcell growth the rotating growth vial may be oscillated at a firstperiodicity (e.g., every 60 seconds), and then a later stage of cellgrowth the rotating growth vial may be oscillated at a secondperiodicity (e.g., every one second) different from the firstperiodicity.

The rotating growth vial 500 may be reusable or, preferably, therotating growth vial is consumable. In some embodiments, the rotatinggrowth vial is consumable and is presented to the user pre-filled withgrowth medium, where the vial is hermetically sealed at the open end 504with a foil seal. A medium-filled rotating growth vial packaged in sucha manner may be part of a kit for use with a stand-alone cell growthdevice or with a cell growth module that is part of an automatedmulti-module cell processing instrument. To introduce cells into thevial, a user need only pipette up a desired volume of cells and use thepipette tip to punch through the foil seal of the vial. Open end 504 mayoptionally include an extended lip 502 to overlap and engage with thecell growth device (not shown). In automated systems, the rotatinggrowth vial 500 may be tagged with a barcode or other identifying meansthat can be read by a scanner or camera that is part of the automatedsystem (not shown).

The volume of the rotating growth vial 500 and the volume of the cellculture (including growth medium) may vary greatly, but the volume ofthe rotating growth vial 500 must be large enough for the cell culturein the growth vial to get proper aeration while the vial is rotating. Inpractice, the volume of the rotating growth vial 500 may range from1-250 ml, 2-100 ml, from 5-80 ml, 10-50 ml, or from 12-35 ml. Likewise,the volume of the cell culture (cells+growth media) should beappropriate to allow proper aeration in the rotating growth vial. Thus,the volume of the cell culture should be approximately 10-85% of thevolume of the growth vial or from 20-60% of the volume of the growthvial. For example, for a 35 ml growth vial, the volume of the cellculture would be from about 4 ml to about 27 ml, or from 7 ml to about21 ml.

The rotating growth vial 500 preferably is fabricated from abio-compatible optically transparent material—or at least the portion ofthe vial comprising the light path(s) is transparent. Additionally,material from which the rotating growth vial is fabricated should beable to be cooled to about 4° C. or lower and heated to about 55° C. orhigher to accommodate both temperature-based cell assays and long-termstorage at low temperatures. Further, the material that is used tofabricate the vial must be able to withstand temperatures up to 55° C.without deformation while spinning. Suitable materials include glass,polyvinyl chloride, polyethylene, polyamide, polyethylene,polypropylene, polycarbonate, poly(methyl methacrylate (PMMA),polysulfone, polyurethane, and co-polymers of these and other polymers.Preferred materials include polypropylene, polycarbonate, orpolystyrene. In some embodiments, the rotating growth vial isinexpensively fabricated by, e.g., injection molding or extrusion.

FIGS. 5B-5D show an embodiment of a cell growth module 550 comprising arotating growth vial 500. FIG. 5B is a perspective view of oneembodiment of a cell growth device 550. FIG. 5C depicts a cut-away viewof the cell growth device 550 from FIG. 5B. In both figures, therotating growth vial 500 is seen positioned inside a main housing 526with the extended lip 502 of the rotating growth vial 500 extendingabove the main housing 526. Additionally, end housings 522, a lowerhousing 532, and flanges 524 are indicated in both figures. Flanges 524are used to attach the cell growth device to heating/cooling means orother structure (not shown). FIG. 5C depicts additional detail. In FIG.5C, upper bearing 542 and lower bearing 530 are shown positioned in mainhousing 526. Upper bearing 542 and lower bearing 530 support thevertical load of rotating growth vial 500. Lower housing 532 containsthe drive motor 536. The cell growth device of FIG. 5C comprises twolight paths: a primary light path 534, and a secondary light path 530.Light path 534 corresponds to light path 510 positioned in theconstricted portion of the tapered-to-constricted portion of therotating growth vial, and light path 530 corresponds to light path 508in the tapered portion of the tapered-to-constricted portion of therotating growth vial. Light paths 510 and 508 are not shown in FIG. 5Cbut may be seen in, e.g., FIG. 5A. In addition to light paths 534 and530, there is an emission board 528 to illuminate the light path(s), anddetector board 546 to detect the light after the light travels throughthe cell culture liquid in the rotating growth vial.

The motor 536 used to rotate the rotating growth vial 500 in someembodiments is a brushless DC type drive motor with built-in drivecontrols that can be set to hold a constant revolution per minute (RPM)between 0 and about 3000 RPM. Alternatively, other motor types such as astepper, servo, brushed DC, and the like can be used. Optionally, themotor 506 may also have direction control to allow reversing of therotational direction, and a tachometer to sense and report actual RPM.The motor is controlled by a processor (not shown) according to, e.g.,standard protocols programmed into the processor and/or user input, andthe motor may be configured to vary RPM to cause axial precession of thecell culture thereby enhancing mixing, e.g., to prevent cellaggregation, increase aeration, and optimize cellular respiration.

Main housing 526, end housings 522 and lower housing 532 of the cellgrowth device 550 may be fabricated from any suitable, robust materialincluding aluminum, stainless steel, and other thermally conductivematerials, including plastics. These structures or portions thereof canbe created through various techniques, e.g., metal fabrication,injection molding, creation of structural layers that are fused, etc.Whereas the rotating growth vial is envisioned in some embodiments to bereusable but preferably is consumable, the other components of the cellgrowth device 550 are preferably reusable and can function as astand-alone benchtop device or, as here, as a module in a multi-modulecell processing system.

The processor (not shown) of the cell growth system may be programmedwith information to be used as a “blank” or control for the growing cellculture. A “blank” or control is a vessel containing cell growth mediumonly, which yields 100% transmittance and 0 OD, while the cell samplewill deflect light rays and will have a lower percent transmittance andhigher OD. As the cells grow in the media and become denser,transmittance will decrease and OD will increase. The processor of thecell growth system may be programmed to use wavelength values for blankscommensurate with the growth media typically used in cell culture(whether, e.g., mammalian cells, bacterial cells, animal cells, yeastcells, etc.). Alternatively, a second spectrophotometer and vessel maybe included in the cell growth system, where the secondspectrophotometer is used to read a blank at designated intervals.

FIG. 5D illustrates a cell growth device as part of an assemblycomprising the cell growth device of FIG. 5B coupled to light source590, detector 592, and thermal components 594. The rotating growth vial500 is inserted into the cell growth device. Components of the lightsource 590 and detector 592 (e.g., such as a photodiode with gaincontrol to cover 5-log) are coupled to the main housing of the cellgrowth device. The lower housing 532 that houses the motor that rotatesthe rotating growth vial is illustrated, as is one of the flanges 524that secures the cell growth device to the assembly. Also illustrated isa Peltier device or thermoelectric cooler 594. In this embodiment,thermal control is accomplished by attachment and electrical integrationof the cell growth device 500 to the thermal device 594 via the flange504 on the base of the lower housing 532. Thermoelectric coolers arecapable of “pumping” heat to either side of a junction, either cooling asurface or heating a surface depending on the direction of current flow.In one embodiment, a thermistor is used to measure the temperature ofthe main housing and then, through a standard electronicproportional-integral-derivative (PID) controller loop, the rotatinggrowth vial 500 is controlled to approximately +/−0.5° C.

In certain embodiments, a rear-mounted power entry module contains thesafety fuses and the on-off switch, which when switched on powers theinternal AC and DC power supplies (not shown) activating the processor.Measurements of optical densities (OD) at programmed time intervals areaccomplished using a 600 nm Light Emitting Diode (LED) (not shown) thathas been columnated through an optic into the lower constricted portionof the rotating growth vial which contains the cells of interest. Thelight continues through a collection optic to the detection system whichconsists of a (digital) gain-controlled silicone photodiode. Generally,optical density is normally shown as the absolute value of the logarithmwith base 10 of the power transmission factors of an optical attenuator:OD=−log 10 (Power out/Power in). Since OD is the measure of opticalattenuation—that is, the sum of absorption, scattering, andreflection—the cell growth device OD measurement records the overallpower transmission, so as the cells grow and become denser in populationthe OD (the loss of signal) increases. The OD system is pre-calibratedagainst OD standards with these values stored in an on-board memoryaccessible by the measurement program.

In use, cells are inoculated (cells can be pipetted, e.g., from anautomated liquid handling system or by a user) into pre-filled growthmedia of a rotating growth vial by piercing though the foil seal. Theprogrammed software of the cell growth device sets the controltemperature for growth, typically 30° C., then slowly starts therotation of the rotating growth vial. The cell/growth media mixtureslowly moves vertically up the wall due to centrifugal force allowingthe rotating growth vial to expose a large surface area of the mixtureto a normal oxygen environment. The growth monitoring system takeseither continuous readings of the OD or OD measurements at pre-set orpre-programmed time intervals. These measurements are stored in internalmemory and if requested the software plots the measurements versus timeto display a growth curve. If enhanced mixing is required, e.g., tooptimize growth conditions, the speed of the vial rotation can be variedto cause an axial precession of the liquid, and/or a completedirectional change can be performed at programmed intervals. The growthmonitoring can be programmed to automatically terminate the growth stageat a pre-determined OD, and then quickly cool the mixture to a lowertemperature to inhibit further growth.

One application for the cell growth device 550 is to constantly measurethe optical density of a growing cell culture. One advantage of thedescribed cell growth device is that optical density can be measuredcontinuously (kinetic monitoring) or at specific time intervals; e.g.,every 5, 10, 15, 20, 30 45, or 60 seconds, or every 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 on minutes. While the cell growth device has been describedin the context of measuring the optical density (OD) of a growing cellculture, it should, however, be understood by a skilled artisan giventhe teachings of the present specification that other cell growthparameters can be measured in addition to or instead of cell culture OD.For example, spectroscopy using visible, UV, or near infrared (NIR)light allows monitoring the concentration of nutrients and/or wastes inthe cell culture. Additionally, spectroscopic measurements may be usedto quantify multiple chemical species simultaneously. Nonsymmetricchemical species may be quantified by identification of characteristicabsorbance features in the NIR. Conversely, symmetric chemical speciescan be readily quantified using Raman spectroscopy. Many criticalmetabolites, such as glucose, glutamine, ammonia, and lactate havedistinct spectral features in the IR, such that they may be easilyquantified. The amount and frequencies of light absorbed by the samplecan be correlated to the type and concentration of chemical speciespresent in the sample. Each of these measurement types provides specificadvantages. FT-NIR provides the greatest light penetration depth and canbe used for thicker sample. FT-mid-IR (MIR) provides information that ismore easily discernible as being specific for certain analytes as thesewavelengths are closer to the fundamental IR absorptions. FT-Raman isadvantageous when interference due to water is to be minimized. Otherspectral properties can be measured via, e.g., dielectric impedencespectroscopy, visible fluorescence, fluorescence polarization, orluminescence. Additionally, the cell growth device may includeadditional sensors for measuring, e.g., dissolved oxygen, carbondioxide, pH, conductivity, and the like.

The Cell Concentration Module

FIGS. 6A-6I depict variations on one embodiment of a cellconcentration/buffer exchange cassette and module that utilizestangential flow filtration. One embodiment of a cell concentrationdevice described herein operates using tangential flow filtration (TFF),also known as crossflow filtration, in which the majority of the feedflows tangentially over the surface of the filter thereby reducing cake(retentate) formation as compared to dead-end filtration, in which thefeed flows into the filter. Secondary flows relative to the main feedare also exploited to generate shear forces that prevent filter cakeformation and membrane fouling thus maximizing particle recovery, asdescribed below.

The TFF device described herein was designed to take into account twoprimary design considerations. First, the geometry of the TFF deviceleads to filtering the cell culture over a large surface area so as tominimize processing time. Second, the design of the TFF device isconfigured to minimize filter fouling. FIG. 6A is a general model 150 oftangential flow filtration. The TFF device operates using tangentialflow filtration, also known as cross-flow filtration. FIG. 6A showscells flowing over a membrane 124, where the feed flow of the cells 152in medium or buffer is parallel to the membrane 124. TFF is differentfrom dead-end filtration where both the feed flow and the pressure dropare perpendicular to a membrane or filter.

FIG. 6B depicts a top view of the lower member of one embodiment of aTFF device/module providing tangential flow filtration. As can be seenin the embodiment of the TFF device of FIG. 6B, TFF device 600 comprisesa channel structure 616 comprising a flow channel 602 b through which acell culture is flowed. The channel structure 616 comprises a singleflow channel 602 b that is horizontally bifurcated by a membrane (notshown) through which buffer or medium may flow, but cells cannot. Thisparticular embodiment comprises an undulating serpentine geometry 614(i.e., the small “wiggles” in the flow channel 602) and a serpentine“zig-zag” pattern where the flow channel 602 crisscrosses the devicefrom one end at the left of the device to the other end at the right ofthe device. The serpentine pattern allows for filtration over a highsurface area relative to the device size and total channel volume, whilethe undulating contribution creates a secondary inertial flow to enableeffective membrane regeneration preventing membrane fouling. Although anundulating geometry and serpentine pattern are exemplified here, otherchannel configurations may be used as long as the channel can bebifurcated by a membrane, and as long as the channel configurationprovides for flow through the TFF module in alternating directions. Inaddition to the flow channel 602 b, portals 604 and 606 as part of thechannel structure 616 can be seen, as well as recesses 608. Portals 604collect cells passing through the channel on one side of a membrane (notshown) (the “retentate”), and portals 606 collect the medium (“filtrate”or “permeate”) passing through the channel on the opposite side of themembrane (not shown). In this embodiment, recesses 608 accommodatescrews or other fasteners (not shown) that allow the components of theTFF device to be secured to one another.

The length 610 and width 612 of the channel structure 616 may varydepending on the volume of the cell culture to be grown and the opticaldensity of the cell culture to be concentrated. The length 610 of thechannel structure 616 typically is from 1 mm to 300 mm, or from 50 mm to250 mm, or from 60 mm to 200 mm, or from 70 mm to 150 mm, or from 80 mmto 100 mm. The width of the channel structure 616 typically is from 1 mmto 120 mm, or from 20 mm to 100 mm, or from 30 mm to 80 mm, or from 40mm to 70 mm, or from 50 mm to 60 mm. The cross-section configuration ofthe flow channel 102 may be round, elliptical, oval, square,rectangular, trapezoidal, or irregular. If square, rectangular, oranother shape with generally straight sides, the cross section may befrom about 10 μm to 1000 μm wide, or from 200 μm to 800 μm wide, or from300 μm to 700 μm wide, or from 400 μm to 600 μm wide; and from about 10μm to 1000 μm high, or from 200 μm to 800 μm high, or from 300 μm to 700μm high, or from 400 μm to 600 μm high. If the cross section of the flowchannel 602 is generally round, oval or elliptical, the radius of thechannel may be from about 50 μm to 1000 μm in hydraulic radius, or from5 μm to 800 μm in hydraulic radius, or from 200 μm to 700 μm inhydraulic radius, or from 300 μm to 600 μm wide in hydraulic radius, orfrom about 200 to 500 μm in hydraulic radius.

When looking at the top view of the TFF device/module of FIG. 6B, notethat there are two retentate portals 604 and two filtrate portals 606,where there is one of each type portal at both ends (e.g., the narrowedge) of the device 600. In other embodiments, retentate and filtrateportals can on the same surface of the same member (e.g., upper or lowermember), or they can be arranged on the side surfaces of the assembly.Unlike other TFF devices that operate continuously, the TFFdevice/module described herein uses an alternating method forconcentrating cells. The overall work flow for cell concentration usingthe TFF device/module involves flowing a cell culture or cell sampletangentially through the channel structure. The membrane bifurcating theflow channels retains the cells on one side of the membrane and allowsunwanted medium or buffer to flow across the membrane into a filtrateside (e.g, lower member 620) of the device. In this process, a fixedvolume of cells in medium or buffer is driven through the device untilthe cell sample is collected into one of the retentate portals 604, andthe medium/buffer that has passed through the membrane is collectedthrough one or both of the filtrate portals 606. All types ofprokaryotic and eukaryotic cells—both adherent and non-adherentcells—can be grown in the TFF device. Adherent cells may be grown onbeads or other cell scaffolds suspended in medium that flow through theTFF device.

In the cell concentration process, passing the cell sample through theTFF device and collecting the cells in one of the retentate portals 604while collecting the medium in one of the filtrate portals 606 isconsidered “one pass” of the cell sample. The transfer between retentatereservoirs “flips” the culture, The retentate and filtrate portalscollecting the cells and medium, respectively, for a given pass resideon the same end of TFF device/module 600 with fluidic connectionsarranged so that there are two distinct flow layers for the retentateand filtrate sides, but if the retentate portal 604 resides on the uppermember of device/module 600 (that is, the cells are driven through thechannel above the membrane and the filtrate (medium) passes to theportion of the channel below the membrane), the filtrate portal 606 willreside on the lower member of device/module 100 and vice versa (that is,if the cell sample is driven through the channel below the membrane, thefiltrate (medium) passes to the portion of the channel above themembrane). This configuration can be seen more clearly in FIGS. 6C-6D,where the retentate flows 660 from the retentate portals 604 and thefiltrate flows 670 from the filtrate portals 606.

At the conclusion of a “pass” in the growth concentration process, thecell sample is collected by passing through the retentate portal 604 andinto the retentate reservoir (not shown). To initiate another “pass”,the cell sample is passed again through the TFF device, this time in aflow direction that is reversed from the first pass. The cell sample iscollected by passing through the retentate portal 604 and into retentatereservoir (not shown) on the opposite end of the device/module from theretentate portal 604 that was used to collect cells during the firstpass. Likewise, the medium/buffer that passes through the membrane onthe second pass is collected through the filtrate portal 606 on theopposite end of the device/module from the filtrate portal 606 that wasused to collect the filtrate during the first pass, or through bothportals. This alternating process of passing the retentate (theconcentrated cell sample) through the device/module is repeated untilthe cells have been concentrated to a desired volume, and both filtrateportals can be open during the passes to reduce operating time. Inaddition, buffer exchange may be effected by adding a desired buffer (orfresh medium) to the cell sample in the retentate reservoir, beforeinitiating another “pass”, and repeating this process until the oldmedium or buffer is diluted and filtered out and the cells reside infresh medium or buffer. Note that buffer exchange and cell concentrationmay (and typically do) take place simultaneously.

FIG. 6C depicts a top view of upper (622) and lower (620) members of anexemplary TFF module. Again, portals 604 and 606 are seen. As notedabove, recesses—such as the recesses 608 seen in FIG. 6B—provide a meansto secure the components (upper member 622, lower member 620, andmembrane 624) of the TFF device/membrane to one another during operationvia, e.g., screws or other like fasteners. However, in alterativeembodiments an adhesive, such as a pressure sensitive adhesive, orultrasonic welding, or solvent bonding, may be used to couple the uppermember 622, lower member 620, and membrane 624 together. Indeed, one ofordinary skill in the art given the guidance of the present disclosurecan find yet other configurations for coupling the components of the TFFdevice, such as e.g., clamps; mated fittings disposed on the upper andlower members; combination of adhesives, welding, solvent bonding, andmated fittings; and other such fasteners and couplings.

Note that there is one retentate portal and one filtrate portal on each“end” (e.g., the narrow edges) of the TFF device/module. The retentateand filtrate portals on the left side of the device/module will collectcells (flow path at 660) and medium (flow path at 670), respectively,for the same pass. Likewise, the retentate and filtrate portals on theright side of the device/module will collect cells (flow path at 660)and medium (flow path at 670), respectively, for the same pass. In thisembodiment, the retentate is collected from portals 604 on the topsurface of the TFF device, and filtrate is collected from portals 606 onthe bottom surface of the device. The cells are maintained in the TFFflow channel above the membrane 624, while the filtrate (medium) flowsthrough membrane 624 and then through portals 606; thus, thetop/retentate portals and bottom/filtrate portals configuration ispractical. It should be recognized, however, that other configurationsof retentate and filtrate portals may be implemented such as positioningboth the retentate and filtrate portals on the side (as opposed to thetop and bottom surfaces) of the TFF device. In FIG. 6C, the channelstructure 602 b can be seen on the bottom member 620 of the TFF device600. However, in other embodiments, retentate and filtrate portals canreside on the same of the TFF device.

Also seen in FIG. 6C is membrane or filter 624. Filters or membranesappropriate for use in the TFF device/module are those that are solventresistant, are contamination free during filtration, and are able toretain the types and sizes of cells of interest. For example, in orderto retain small cell types such as bacterial cells, pore sizes can be aslow as 0.2 μm, however for other cell types, the pore sizes can be ashigh as 5 μm. Indeed, the pore sizes useful in the TFF device/moduleinclude filters with sizes from 0.20 μm, 0.21 μm, 0.22 μm, 0.23 μm, 0.24μm, 0.25 μm, 0.26 μm, 0.27 μm, 0.28 μm, 0.29 μm, 0.30 μm, 0.31 μm, 0.32μm, 0.33 μm, 0.34 μm, 0.35 μm, 0.36 μm, 0.37 μm, 0.38 μm, 0.39 μm, 0.40μm, 0.41 μm, 0.42 μm, 0.43 μm, 0.44 μm, 0.45 μm, 0.46 μm, 0.47 μm, 0.48μm, 0.49 μm, 0.50 μm and larger. The filters may be fabricated from anysuitable non-reactive material including cellulose mixed ester(cellulose nitrate and acetate) (CME), polycarbonate (PC),polyvinylidene fluoride (PVDF), polyethersulfone (PES),polytetrafluoroethylene (PTFE), nylon, glass fiber, or metal substratesas in the case of laser or electrochemical etching. The TFF device shownin FIGS. 6C and 6D do not show a seat in the upper 612 and lower 620members where the filter 624 can be seated or secured (for example, aseat half the thickness of the filter in each of upper 612 and lower 620members); however, such a seat is contemplated in some embodiments.

FIG. 6D depicts a bottom view of upper and lower components of theexemplary TFF module shown in FIG. 6C. FIG. 6D depicts a bottom view ofupper (622) and lower (620) components of an exemplary TFF module. Againportals 604 and 606 are seen. Note again that there is one retentateportal and one filtrate portal on each end of the device/module. Theretentate and filtrate portals on the left side of the device/modulewill collect cells (flow path at 660) and medium (flow path at 670),respectively, for the same pass. Likewise, the retentate and filtrateportals on the right side of the device/module will collect cells (flowpath at 660) and medium (flow path at 670), respectively, for the samepass. In FIG. 6D, the channel structure 602 a can be seen on the uppermember 622 of the TFF device 600. Thus, looking at FIGS. 6C and 6D, notethat there is a channel structure 602 (602 a and 602 b) in both theupper and lower members, with a membrane 624 between the upper and lowerportions of the channel structure. The channel structure 602 of theupper 622 and lower 620 members (602 a and 602 b, respectively) mate tocreate the flow channel with the membrane 624 positioned horizontallybetween the upper and lower members of the flow channel therebybifurcating the flow channel.

Medium exchange (during cell growth) or buffer exchange (during cellconcentration or rendering the cells competent) is performed on the TFFdevice/module by adding fresh medium to growing cells or a desiredbuffer to the cells concentrated to a desired volume; for example, afterthe cells have been concentrated at least 20-fold, 30-fold, 40-fold,50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold,200-fold or more. A desired exchange medium or exchange buffer is addedto the cells either by addition to the retentate reservoir or thoroughthe membrane from the filtrate side and the process of passing the cellsthrough the TFF device 600 is repeated until the cells have been grownto a desired optical density or concentrated to a desired volume in theexchange medium or buffer. This process can be repeated any number ofdesired times so as to achieve a desired level of exchange of the bufferand a desired volume of cells. The exchange buffer may comprise, e.g.,glycerol or sorbitol thereby rendering the cells competent fortransformation in addition to decreasing the overall volume of the cellsample.

The TFF device 600 may be fabricated from any robust material in whichchannels (and channel branches) may be milled including stainless steel,silicon, glass, aluminum, or plastics including cyclic-olefin copolymer(COC), cyclo-olefin polymer (COP), polystyrene, polyvinyl chloride,polyethylene, polyamide, polyethylene, polypropylene, acrylonitrilebutadiene, polycarbonate, polyetheretheketone (PEEK), poly(methylmethylacrylate) (PMMA), polysulfone, and polyurethane, and co-polymersof these and other polymers. If the TFF device/module is disposable,preferably it is made of plastic. In some embodiments, the material usedto fabricate the TFF device/module is thermally-conductive so that thecell culture may be heated or cooled to a desired temperature. Incertain embodiments, the TFF device is formed by precision mechanicalmachining, laser machining, electro discharge machining (for metaldevices); wet or dry etching (for silicon devices); dry or wet etching,powder or sandblasting, photostructuring (for glass devices); orthermoforming, injection molding, hot embossing, or laser machining (forplastic devices) using the materials mentioned above that are amenableto this mass production techniques.

FIG. 6E depicts an exemplary configuration of an assembled TFF device,where, like the other configurations, the upper member and lower memberin combination form a channel structure with a membrane disposed betweenthe upper and lower members; however, in this configuration in additionto the retentate reservoirs, there is in addition an optional buffer ormedium reservoir positioned between the retentate reservoirs, and alower filtrate or permeate reservoir. In the TFF device 6000configuration shown in FIG. 6E, 6044 is the top or cover of the TFFdevice 6000, having three ports 6046, where there is a pipette tip 6048disposed in the right-most port 6046. The top 6044 of the TFF device6000 is adjacent to and in operation is coupled with a combinedreservoir and upper member structure 6050. Combined reservoir and uppermember structure 6050 comprises a top surface that is adjacent the topor cover 6044 of the TFF device, a bottom surface which comprises theupper member 6022 of the TFF device, where the upper member 6022 of theTFF device defines the upper portion of the flow channel (not shown)disposed on the bottom surface of the upper member 6022 of the combinedreservoir and upper member structure 6050. Additionally, combinedreservoir and upper member structure 6050 comprises two retentatereservoirs 6080 and an optional buffer or medium reservoir 6082. Theretentate reservoirs are fluidically coupled to the upper portion of theflow channel, and the buffer or medium reservoir is fluidically coupledto the retentate reservoirs. Also seen in this assembled view of TFFdevice 6000 is membrane 6024, lower member 6020 which, as describedpreviously comprises on its top surface the lower portion of thetangential flow channel (not shown), where the channel structures of theupper member 6022 and lower member 6020 (neither shown in this view)mate to form a single flow channel. Beneath and adjacent to lower member6020 is a gasket 6040, which is interposed between lower member 6020 andan optional filtrate (or permeate) reservoir 6042. The filtratereservoir 6042 is in fluid connection with the lower portion of the flowchannel, as a receptacle for the filtrate or permeate that is removedfrom the cell culture. In operation, top 6044, combined reservoir andupper member structure 6050, membrane 6024, lower member 6020, gasket6040, and filtrate reservoir 6042 are coupled and secured together to befluid- and air-tight. The assembled TFF device 1100 typically is from 4to 25 cm in height, or from 5 to 20 cm in height, or from 7 to 15 cm inheight; from 5 to 30 cm in length, or from 8 to 25 cm in length, or from10 to 20 cm in length; and is from 3 to 15 cm in depth, or from 5 to 10cm in depth. An exemplary TFF device is 11 cm in height, 12 cm inlength, and 8 cm in depth. The retentate reservoirs, buffer or mediumreservoir, and tangential flow channel-forming structures may beconfigured to be cooled to 4° C. for cell maintenance. The dimensionsfor the serpentine channel recited above, as well as the specificationsand materials for the filter and the TFF device apply to the embodimentof the device shown in FIGS. 6E-6I. In embodiments including the presentembodiment, up to 120 mL of cell culture can be grown and/or filtered,or up to 100 mL, 90 mL, 80 mL, 70 mL, 60 mL, 50 mL, 40 mL, 30 mL or 20mL of cell culture can be grown and/or filtered.

FIG. 6F depicts an exploded perspective view of TFF device 6000. In thisconfiguration, 6044 is the top or cover of the TFF device 6000, havingthree ports 6046, where there is a pipette tip 6048 disposed in theleft-most port 6046. The top 6044 of the TFF device 6000 is, inoperation, coupled with a combined reservoir and upper member structure6050. Combined reservoir and upper member structure 6050 comprises a topsurface that, in operation, is adjacent the top or cover 6044 of the TFFdevice, a bottom surface which comprises the upper member 6022 of theTFF device, where the upper member 6022 of the TFF device defines theupper portion of the tangential flow channel (not shown). Combinedreservoir and upper member structure 6050 comprises two retentatereservoirs 6080 and an optional buffer or medium reservoir 6082. Theretentate reservoirs are fluidically coupled to the upper portion of theflow channel, and the optional buffer or medium reservoir is fluidicallycoupled to the retentate reservoirs. Also seen in this exploded view ofTFF device 6000 is lower member 6020 which, as described previouslycomprises on its top surface the lower portion of the tangential flowchannel 6002 b (seen on the top surface of lower member 6020), where theupper and lower portions of the channel structures of the upper member6022 and lower member 6020, respectively, when coupled mate to form asingle flow channel (the membrane that is interposed between the uppermember 6022 and lower member 6020 in operation is not shown). Beneathlower member 6020 is gasket 6040, which in operation is interposedbetween lower member 6020 and a filtrate (or permeate) reservoir 6042.In operation, top 6044, combined reservoir and upper member structure6050, membrane (not shown), lower member 6020, gasket 6040, and filtratereservoir 6042 are coupled and secured together to be fluid- andair-tight. In FIG. 6F, fasteners are shown that can be used to couplethe various structures (top 6044, combined reservoir and upper memberstructure 6050, membrane (not shown), lower member 6020, gasket 6040,and filtrate reservoir 6042) together. However, as an alternative toscrews or other like fasteners, the various structures of TFF device6000 can be coupled using an adhesive, such as a pressure sensitiveadhesive; ultrasonic welding; or solvent bonding. Further, a combinationof fasteners, adhesives, and/or welding types may be employed to couplethe various structures of the TFF device. One of ordinary skill in theart given the guidance of the present disclosure could find yet otherconfigurations for coupling the components of TFF device 6000, such ase.g., clamps, mated fittings, and other such fasteners.

FIG. 6G depicts combined reservoir and upper member structure 6050,comprising two retentate reservoirs 6080 and an optional buffer ormedium reservoir 6082, as well as upper member 6020, which is disposedon the bottom of combined reservoir and upper member structure 6050.Upper member 6022 of the TFF device defines the upper portion of thetangential flow channel (not shown) disposed on the bottom surface ofthe combined reservoir and upper member structure 6050. FIG. 6H is atop-down view of the upper surface of combined reservoir and uppermember structure 6050, depicting the top of retentate reservoirs 6080and buffer or medium reservoir 6082, as well as fluid or vacuum ports6046. The retentate reservoirs are fluidically coupled to the upperportion of the flow channel, and the buffer or medium reservoir isfluidically coupled to the retentate reservoirs. FIG. 6I is a bottom-upview of the lower surface of combined reservoir and upper memberstructure 6050, showing the upper member 6020 with the upper portion ofthe tangential flow channel 6002 a disposed on the bottom surface ofupper member 6020. The flow channel 6002 a disposed on the bottomsurface of upper member 6020 in operation is mated to the bottom portionof the tangential flow channel disposed on the top surface of the lowermember (not shown in this view, but see FIG. 6F), where the upper andlower portions of the flow channel structure mate to form a single flowchannel.

As an alternative to the TFF module described above, a cellconcentration module comprising a hollow filter may be employed.Examples of filters suitable for use in the present invention includemembrane filters, ceramic filters and metal filters. The filter may beused in any shape; the filter may for example be cylindrical oressentially flat. Preferably, the filter used is a membrane filter,preferably a hollow fiber filter. The term “hollow fiber” is meant atubular membrane. The internal diameter of the tube is at least 0.1 mm,more preferably at least 0.5 mm, most preferably at least 0.75 mm andpreferably the internal diameter of the tube is at most 10 mm, morepreferably at most 6 mm, most preferably at most 1 mm. Filter modulescomprising hollow fibers are commercially available from variouscompanies, including G.E. Life Sciences (Marlborough, Mass.) andInnovaPrep (Drexel, Mo.). Specific examples of hollow fiber filtersystems that can be used, modified or adapted for use in the presentmethods and systems include, but are not limited to, U.S. Pat. Nos.9,738,918; 9,593,359; 9,574,977; 9,534,989; 9,446,354; 9,295,824;8,956,880; 8,758,623; 8,726,744; 8,677,839; 8,677,840; 8,584,536;8,584,535; and 8,110,112.

Nucleic Acid Assembly Module

Certain embodiments of the automated multi-module cell editinginstruments of the present disclosure optionally include a nucleic acidassembly module. The nucleic acid assembly module is configured toaccept and assemble the nucleic acids necessary to facilitate thedesired genome editing events. In general, the term “vector” refers to anucleic acid molecule capable of transporting a desired nucleic acid towhich it has been linked into a cell. Vectors include, but are notlimited to, nucleic acid molecules that are single-stranded,double-stranded, or partially double-stranded; nucleic acid moleculesthat include one or more free ends, no free ends (e.g., circular);nucleic acid molecules that include DNA, RNA, or both; and othervarieties of polynucleotides known in the art. One type of vector is a“plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,where virally-derived DNA or RNA sequences are present in the vector forpackaging into a virus (e.g. retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses). Viral vectors also include polynucleotidescarried by a virus for transfection into a host cell. Certain vectorsare capable of autonomous replication in a host cell into which they areintroduced (e.g. bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively-linked.Such vectors are referred to herein as “expression vectors” or “editingvectors.” Common expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. Additional vectors includefosmids, phagemids, and synthetic chromosomes.

Recombinant expression vectors can include a nucleic acid in a formsuitable for transcription, and for some nucleic acid sequences,translation and expression of the nucleic acid in a host cell, whichmeans that the recombinant expression vectors include one or moreregulatory elements—which may be selected on the basis of the host cellsto be used for expression—that are operatively-linked to the nucleicacid sequence to be expressed. Within a recombinant expression vector,“operably linked” is intended to mean that the nucleotide sequence ofinterest is linked to the regulatory element(s) in a manner that allowsfor transcription, and for some nucleic acid sequences, translation andexpression of the nucleotide sequence (e.g. in an in vitrotranscription/translation system or in a host cell when the vector isintroduced into the host cell). Appropriate recombination and cloningmethods are disclosed in US Pub. No. 2004/0171156, the contents of whichare herein incorporated by reference in their entirety for all purposes.

In some embodiments, a regulatory element is operably linked to one ormore elements of a targetable nuclease system so as to drivetranscription, and for some nucleic acid sequences, translation andexpression of the one or more components of the targetable nucleasesystem.

In addition, the polynucleotide sequence encoding the nucleicacid-guided nuclease can be codon optimized for expression in particularcells, such as prokaryotic or eukaryotic cells. Eukaryotic cells can beyeast, fungi, algae, plant, animal, or human cells. Eukaryotic cells maybe those of or derived from a particular organism, such as a mammal,including but not limited to human, mouse, rat, rabbit, dog, ornon-human mammal including non-human primate. In addition oralternatively, a vector may include a regulatory element operably likedto a polynucleotide sequence, which, when transcribed, forms a guideRNA.

The nucleic acid assembly module can be configured to perform a widevariety of different nucleic acid assembly techniques in an automatedfashion. Nucleic acid assembly techniques that can be performed in thenucleic acid assembly module of the disclosed automated multi-modulecell editing instruments include, but are not limited to, those assemblymethods that use restriction endonucleases, including PCR, BioBrickassembly (U.S. Pat. No. 9,361,427), Type IIS cloning (e.g., GoldenGateassembly, European Patent Application Publication EP 2 395 087 A1), andLigase Cycling Reaction (de Kok, ACS Synth Biol., 3(2):97-106 (2014);Engler, et al., PLoS One, 3(11):e3647 (2008); and U.S. Pat. No.6,143,527). In other embodiments, the nucleic acid assembly techniquesperformed by the disclosed automated multi-module cell editinginstruments are based on overlaps between adjacent parts of the nucleicacids, such as Gibson Assembly®, CPEC, SLIC, Ligase Cycling etc.Additional assembly methods include gap repair in yeast (Bessa, Yeast,29(10):419-23 (2012)), gateway cloning (Ohtsuka, Curr Pharm Biotechnol,10(2):244-51 (2009)); U.S. Pat. Nos. 5,888,732; and 6,277,608), andtopoisomerase-mediated cloning (Udo, PLoS One, 10(9):e0139349 (2015);and U.S. Pat. No. 6,916,632). These and other nucleic acid assemblytechniques are described, e.g., in Sands and Brent, Curr Protoc MolBiol., 113:3.26.1-3.26.20 (2016).

The nucleic acid assembly module is temperature controlled dependingupon the type of nucleic acid assembly used in the automatedmulti-module cell editing instrument. For example, when PCR is utilizedin the nucleic acid assembly module, the module includes a thermocyclingcapability allowing the temperatures to cycle between denaturation,annealing and extension steps. When single temperature assembly methods(e.g., isothermal assembly methods) are utilized in the nucleic acidassembly module, the module provides the ability to reach and hold atthe temperature that optimizes the specific assembly process beingperformed. These temperatures and the duration for maintaining thesetemperatures can be determined by a preprogrammed set of parametersexecuted by a script, or manually controlled by the user using theprocessing system of the automated multi-module cell editing instrument.

In one embodiment, the nucleic acid assembly module is a module toperform assembly using a single, isothermal reaction. Certain isothermalassembly methods can combine simultaneously up to 15 nucleic acidfragments based on sequence identity. The assembly method provides, insome embodiments, nucleic acids to be assembled which include anapproximate 20-40 base overlap with adjacent nucleic acid fragments. Thefragments are mixed with a cocktail of three enzymes—an exonuclease, apolymerase, and a ligase-along with buffer components. Because theprocess is isothermal and can be performed in a 1-step or 2-step methodusing a single reaction vessel, isothermal assembly reactions are idealfor use in an automated multi-module cell editing instrument. The 1-stepmethod allows for the assembly of up to five different fragments using asingle step isothermal process. The fragments and the master mix ofenzymes are combined and incubated at 50° C. for up to one hour. For thecreation of more complex constructs with up to fifteen fragments or forincorporating fragments from 100 bp up to 10 kb, typically the 2-step isused, where the 2-step reaction requires two separate additions ofmaster mix; one for the exonuclease and annealing step and a second forthe polymerase and ligation steps.

The Cell Transformation Module

In addition to the modules for cell growth, cell concentration, andnucleic acid assembly, FIGS. 7A-7E depict variations on one embodimentof a cell transformation module (in this case, a flow throughelectroporation device) that may be included in a cellgrowth/concentration/transformation instrument. FIGS. 7A and 7B are topperspective and bottom perspective views, respectively, of six co-joinedflow-through electroporation devices 750. FIG. 7A depicts sixflow-through electroporation units 750 arranged on a single substrate756. Each of the six flow-through electroporation units 750 have wells752 that define cell sample inlets and wells 754 that define cell sampleoutlets. FIG. 7B is a bottom perspective view of the six co-joinedflow-through electroporation devices of FIG. 7A also depicting sixflow-through electroporation units 750 arranged on a single substrate4156. Six inlet wells 4152 can be seen, one for each flow-throughelectroporation unit 750, and one outlet well 754 can be seen (theoutlet well of the left-most flow-through electroporation unit 750).Additionally seen in FIG. 7B are an inlet 702, outlet 704, flow channel706 and two electrodes 708 on either side of a constriction in flowchannel 706 in each flow-through electroporation unit 750. Once the sixflow-through electroporation units 750 are fabricated, they can beseparated from one another (e.g., “snapped apart”) and used one at atime, or alternatively in embodiments two or more flow-throughelectroporation units 750 can be used in parallel without separation.

The flow-through electroporation devices achieve high efficiency cellelectroporation with low toxicity. The flow-through electroporationdevices of the disclosure allow for particularly easy integration withrobotic liquid handling instrumentation that is typically used inautomated systems such as air displacement pipettors. Such automatedinstrumentation includes, but is not limited to, off-the-shelf automatedliquid handling systems from Tecan (Mannedorf, Switzerland), Hamilton(Reno, Nev.), Beckman Coulter (Fort Collins, Colo.), etc.

Generally speaking, microfluidic electroporation—using cell suspensionvolumes of less than approximately 10 ml and as low as 1 μl—allows moreprecise control over a transfection or transformation process andpermits flexible integration with other cell processing tools comparedto bench-scale electroporation devices. Microfluidic electroporationthus provides unique advantages for, e.g., single cell transformation,processing and analysis; multi-unit electroporation deviceconfigurations; and integrated, automatic, multi-module cell processingand analysis.

In specific embodiments of the flow-through electroporation devices ofthe disclosure the toxicity level of the transformation results ingreater than 10% viable cells after electroporation, preferably greaterthan 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%,85%, 90%, or even 95% viable cells following transformation, dependingon the cell type and the nucleic acids being introduced into the cells.

The flow-through electroporation device described in relation to FIGS.7A-7E comprises a housing with an electroporation chamber, a firstelectrode and a second electrode configured to engage with an electricpulse generator, by which electrical contacts engage with the electrodesof the electroporation device. In certain embodiments, theelectroporation devices are autoclavable and/or disposable, and may bepackaged with reagents in a reagent cartridge. The electroporationdevice may be configured to electroporate cell sample volumes between 1μl to 2 ml, 10 μl to 1 ml, 25 μl to 750 μl, or 50 μl to 500 μl. Thecells that may be electroporated with the disclosed electroporationdevices include mammalian cells (including human cells), plant cells,yeasts, other eukaryotic cells, bacteria, archaea, and other cell types.

In one exemplary embodiment, FIG. 7C depicts a top view of aflow-through electroporation device 750 having an inlet 702 forintroduction of cells and an exogenous reagent to be electroporated intothe cells (“cell sample”) and an outlet 704 for the cell samplefollowing electroporation. Electrodes 708 are introduced throughelectrode channels (not shown) in the device. FIG. 7D shows a cutawayview from the top of flow-through electroporation device 750, with theinlet 702, outlet 704, and electrodes 708 positioned with respect to aconstriction in flow channel 706. A side cutaway view of the bottomportion of flow-through electroporation device 750 in FIG. 7Eillustrates that electrodes 708 in this embodiment are positioned inelectrode channels 710 and perpendicular to flow channel 706 such thatthe cell sample flows from the inlet channel 712 through the flowchannel 706 to the outlet channel 714, and in the process the cellsample flows into the electrode channels 710 to be in contact withelectrodes 708. In this aspect, the inlet channel, outlet channel andelectrode channels all originate from the top planar side of the device;however, the flow-through electroporation architecture depicted in FIGS.7C-7E is but one architecture useful with the reagent cartridgesdescribed herein. Additional electrode architectures are described,e.g., in U.S. Ser. No. 16/147,120, filed 24 Sep. 2018; Ser. No.16/147,865, filed 30 Sep. 2018; and Ser. No. 16/147,871, filed 30 Sep.2018.

The Cell Enrichment Module

One optional aspect provides automated modules and instruments fornucleic acid-guided nuclease genome editing that implement enrichmenttechniques for cells whose genomes have been properly edited. Theenrichment modules perform methods that use cell singulation andnormalization to reduce growth competition between edited and uneditedcells. Singulation overcomes growth bias from unedited cells or cellscontaining edits conferring growth advantages or disadvantages. Themethods, modules and instruments may be applied to all cell typesincluding, archaeal, prokaryotic, and eukaryotic (e.g., yeast, fungal,plant and animal) cells.

Singulating, optional induction of editing, and normalization of cellcolonies leads to 2-250×, 10-225×, 25-200×, 40-175×, 50-150×, 60-100×,or 5-100× gains in identifying edited cells over, prior art methods andprovides new approaches for generating arrayed or pooled edited cellscomprising genome libraries. Additionally, the methods, modules, andinstruments may be leveraged to create iterative editing systems togenerate combinatorial libraries, identify rare cell edits, and enablehigh-throughput enrichment applications to identify editing activity.

The compositions and methods described herein improve nucleicacid-guided nuclease editing systems in which nucleic acid-guidednucleases (e.g., RNA-guided nucleases) are used to edit specific targetregions in an organism's genome. FIG. 8A depicts a solid wall device 850and a workflow for singulating cells in microwells in the solid walldevice, where in this workflow one or both of the gRNA and nuclease areunder the control of an inducible promoter. At the top left of thefigure (i), there is depicted solid wall device 850 with microwells 852.A section 854 of substrate 850 is shown at (ii), also depictingmicrowells 852. At (iii), a side cross-section of solid wall device 850is shown, and microwells 852 have been loaded, where, in thisembodiment, Poisson loading has taken place; that is, each microwell hasone or no cells, and the likelihood that any one microwell has more thanone cell is low. At (iv), workflow 840 is illustrated where substrate850 having microwells 852 shows microwells 856 with one cell permicrowell, microwells 857 with no cells in the microwells, and onemicrowell 260 with two cells in the microwell. In step 851, and thecells in the microwells are allowed to double approximately 2-50 timesto form clonal colonies (v), then editing is induced 853 by heating thesubstrate (e.g., for temperature-induced editing) or flowing chemicalsunder or over the substrate (e.g., sugars, antibiotics forchemical-induced editing) or by moving the solid wall device to adifferent medium; particularly facile if the solid wall device is placedon a fluid permeable membrane which forms the bottom of microwells 852.After induction of editing 853, many cells in the colonies of cells thathave been edited die as a result of the double-strand cuts caused byactive editing, and there is possibly a lag in growth for the editedcells that do survive but must repair and recover following editing(microwells 858), where cells that do not undergo editing thrive(microwells 859) (vi). All cells are allowed to grow to continue toestablish colonies and normalize, where the colonies of edited cells inmicrowells 858 catch up in size and/or cell number with the cells inmicrowells 859 that do not undergo editing (vii) due to cell senescenceas the unedited cells reach stationary phase. Once the cell colonies arenormalized, either pooling of all cells in the microwells can takeplace, in which case the cells are enriched for edited cells byeliminating the bias from non-editing cells and fitness effects fromediting; alternatively, colony growth in the microwells is monitoredafter editing, and slow growing colonies (e.g., the cells in microwells858) are identified and selected (e.g., “cherry picked”) resulting ineven greater enrichment of edited cells.

In growing the cells, the medium used will depend, of course, on thetype of cells being edited—e.g., bacterial, yeast or mammalian. Forexample, medium for bacterial growth includes LB, SOC, M9 Minimalmedium, and Magic medium; medium for yeast cell growth includes TPD,YPG, YPAD, and synthetic minimal medium; and medium for mammalian cellgrowth includes MEM, DMEM, IMDM, RPMI, and Hanks.

FIG. 8B is a photograph of one embodiment of a solid wall devicecomprising microwells for singulating cells. As can be seen from thephoto, the solid wall device is approximately 2 inches (˜47 mm) indiameter. The solid device seen in this photograph is essentially aperforated disk of 816 stainless steel, where the perforations form thewalls of the microwells, and a filter or membrane is used to form thebottom of the microwells. Use of a filter or membrane (such as a 0.22μPVDF Duropore™ woven membrane filter) allows for medium and/or nutrientsto enter the microwells but prevents the cells from flowing down and outof the microwells. Filter or membrane members that may be used in thesolid wall singulation/growth/editing/normalization devices and modulesare those that are solvent resistant, are contamination free duringfiltration, and are able to retain the types and sizes of cells ofinterest. For example, in order to retain small cell types such asbacterial cells, pore sizes can be as low as 0.2 μm, however for othercell types, the pore sizes can be as high as 0.5 μm. Indeed, the poresizes useful in the cell concentration device/module include filterswith sizes from 0.20 μm, 0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm,0.26 μm, 0.27 μm, 0.28 μm, 0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm,0.34 μm, 0.35 μm, 0.36 μm, 0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm,0.42 μm, 0.43 μm, 0.44 μm, 0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm,0.50 μm and larger. The filters may be fabricated from any suitablematerial including cellulose mixed ester (cellulose nitrate and acetate)(CME), polycarbonate (PC), polyvinylidene fluoride (PVDF),polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon, or glassfiber.

In the photograph shown in FIG. 8B, the perforations are approximately152 nM in diameter, resulting in the microwells having a volume ofapproximately 2.5 nL, with a total of approximately 30,000 wells. Thedistance between the microwells is approximately 279 nMcenter-to-center. Though here the microwells have a volume ofapproximately 2.5 nL, the volume of the microwells may be from 1 to 25nL, or preferably from 2 to 10 nL, and even more preferably from 2 to 4nL. The preferred size/volume of the microwells will depend of cell type(e.g., bacterial, yeast, mammalian). The perforated disk shown here ismade of 316 stainless steel; however other bio-compatible metals andmaterials may be used. The solid wall device may be disposable or it maybe reusable. The solid wall device shown in FIG. 8B is round, but can beof any shape, for example, square, rectangular, oval, etc. Round solidwall devices are useful if petri dishes are used to supply the solidwall device with nutrients via solid medium. The filters used to formthe bottom of the wells of the solid wall device include 0.22μ PVDFDuropore™ woven membrane filters. Further, though a 2-inch (˜47 mm)diameter solid wall device is shown, the solid wall devices may besmaller or larger as desired and the configuration of the solid walldevice will depend on how nutrients are supplied to the solid walldevice, and how media exchange is performed. Although a round solid walldevice is described here, the solid wall devices can be of any shape andsize, including rectangular solid wall devices with 100K, 200K or morewells, in addition to configurations of solid wall devices and cassettesthat are multiplexed, e.g., stacked.

FIGS. 8C-8E are photographs of E. coli cells at low, medium and highmagnification, respectively, singulated via Poisson distribution inmicrowells in a solid wall device with a membrane bottom. FIG. 8C showsdigital growth at low magnification where the darker microwells aremicrowells where cells are growing. FIG. 8D is a top view of microwellsin a solid wall device where the darker microwells are microwells wherecells are growing. FIG. 8E is a photograph of microwells where themembrane (e.g., the permeable membrane that forms the bottom of themicrowells) has been removed, where unpatterned (smooth) microwells aremicrowells where cells are not growing, and microwells with irregularpigment/patterned are microwells where cells are growing, and, in thisphotograph, have filled the microwells in which they are growing. Inthese photographs, a 0.2 μm filter (membrane) was pressed against theperforated metal sold wall device such as the round solid wall devicedepicted in FIG. 8B. The perforated metal solid wall device formed thewalls of the microwells, and the 0.2 μm filter formed the bottom of themicrowells. To load the solid wall device, the E. coli cells were pulledinto the microwells using a vacuum. The solid wall device+filter wasthen placed on an LB agar plate membrane-side down, and the cells weregrown overnight at 30° C., then two days at room temperature. Themembrane was removed and the bottomless microwells were photographed bylight microscopy. Note the ease with which different selective media canbe used to select for certain cell phenotypes; that is, one need onlytransfer the solid wall device+filter to a different plate or petri dishcomprising a desired selective medium or flow a desired selective mediuminto a substrate onto which the solid wall device and coupled membraneare positioned.

In addition to the solid wall cell singulation device described inrelation to FIGS. 8A-8E, other cell singulation devices may be employedin the multi-module cell processing instrument, such as those describedin U.S. Ser. No. 62/735,365, entitled “Detection of Nuclease EditedSequences in Automated Modules and Systems”, filed 24 Sep. 2018, andU.S. Ser. No. 62/781,112, entitled “Improved Detection of NucleaseEdited Sequences in Automated Modules and Systems”, filed 18 Dec. 2018,including singulation by plating on agar, singulation by isolating cellson functionalized islands, singulation within aqueous droplets carriedin a hydrophobic carrier fluid, or singulation within a polymerizedalginate scaffold (for this embodiment of singulation, also see U.S.Ser. No. 62/769,805, entitled “Improved Detection of Nuclease EditedSequences in Automated Modules and Instruments via Bulk Cell Culture”,filed 20 Nov. 2018.

As an alternative to singulation, inducing editing via an induciblepromoter driving one or both of the gRNA and the nuclease at a specifictime in the cell growth cycle may be employed. FIG. 8F shows asimplified flow chart for exemplary methods 8000 for enriching foredited cells. Looking at FIG. 8F, method 8000 begins by designing andsynthesizing editing cassettes 8002. As described in relation to nucleicacid-guided editing above, each editing cassette typically comprises agRNA, a donor DNA, and a PAM or spacer mutation. Once the individualediting cassettes have been synthesized, the individual editingcassettes may be “linked” or “assembled” together and are amplified andassembled into editing vector backbones 8004. The editing vectorscomprising the editing cassettes are then used to transform cells 8006thereby creating a library of transformed cells. In addition to thevectors comprising the assembled editing cassettes, the cells may betransformed simultaneously with a separate engine vector comprising acoding sequence for a nuclease. Alternatively, the cells may already beexpressing the nuclease (e.g., the cells may have already beentransformed with an engine vector or the coding sequence for thenuclease may be stably integrated into the cellular genome) such thatonly the editing vector needs to be transformed into the cells; or thecells may be transformed with a single vector comprising all componentsrequired to perform nucleic acid-guided nuclease genome editing (e.g.,all of the nuclease and an editing cassette), which is advantageous whenemploying curing and recursive rounds of editing.

A variety of delivery systems may be used to introduce (e.g., transformor transfect) nucleic acid-guided nuclease editing system componentsinto a host cell 8008. These delivery systems include the use of yeastsystems, lipofection systems, microinjection systems, biolistic systems,virosomes, liposomes, immunoliposomes, polycations, lipid:nucleic acidconjugates, virions, artificial virions, viral vectors, electroporation,cell permeable peptides, nanoparticles, nanowires, exosomes.Alternatively, molecular trojan horse liposomes may be used to delivernucleic acid-guided nuclease components across the blood brain barrier.Of particular interest is the use of electroporation, particularlyflow-through electroporation (either as a stand-alone instrument or as amodule in an automated multi-module system) as described in, e.g., U.S.Ser. No. 16/024,831 filed 30 Jun. 2018; Ser. No. 16/024,816 filed 30Jun. 2018; Ser. No. 16/147,353 filed 28 Sep. 2018; Ser. No. 16/147,865filed 30 Sep. 2018; and Ser. No. 16/147,871 filed 30 Jun. 2018. If thescreening/selection module is one module in an automated multi-modulecell editing system, the cells are likely transformed in an automatedcell transformation module.

Once transformed 8006, the cells can then be subjected to selectionusing a selectable marker 8008. Selectable markers are employed toselect for cells that have received both the engine and editing vectors,or for cells that have been transformed with a single, combined engineand editing vector. Commonly used selectable markers include drugselectable markers such as ampicillin/carbenicillin, kanamycin,chloramphenicol, erythromycin, tetracycline, gentamicin, bleomycin,streptomycin, rhamnose, puromycin, hygromycin, blasticidin, and G418.

Once cells that have been properly transformed are selected 8008, thenext step in method 8000 is to grow cells in liquid medium until thecells enter (or are close to entering) the stationary phase of growth.Once the cells are in stationary phase 8010 (or nearly so), editing isinduced 8012 in the cells by induction of transcription of one or bothof the nuclease and gRNA. Once editing is induced 8012, the cells can begrown, rendered electrocompetent, and subjected to another round ofediting 8014.

FIG. 8G depicts a typical growth curve 8020 for cells in culture(optical density versus time). Initially there is a lag phase 8022, thenthe cells enter log phase 8024 where they grow quickly, and finally thecells reach stationary phase 8028 where the cells are no longerdividing. The present methods employ inducing transcription of either orboth the nuclease and/or gRNA at timepoint 8026 or later when the cellsare in the stationary phase of growth or nearly so; that is, the cellsare induced at a timepoint at least 60% into the log phase of growth, orat least 65% into the log phase of growth, or at least 70% into the logphase of growth, or at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 79, 98, or99% into the log phase of growth, and at any time during the stationaryphase of growth.

FIG. 8H depicts an exemplary protocol 8050 for performing nucleicacid-guided nuclease genome editing. FIG. 8H depicts the protocols shownin FIG. 8F for editing cells. First, a library or collection of editingvectors 8052 (editing vectors each comprising an editing cassette) isintroduced 8053 (e.g., electroporated) into cultured cells 8054 thatcomprise a coding sequence for a nuclease under the control of aconstitutive or inducible promoter (preferably an inducible promoter),contained 1) on an “engine plasmid” (most often along with a selectablemarker) that has already been transformed into the cells; 2) integratedinto the genome of the cells being transformed; or 3) the codingsequence for the nuclease may be located on the editing vector. Theediting vectors 8052 comprise a donor DNA, a PAM or spacer-alteringsequence (most often a sequence that disables the PAM at the target sitein the genome), a coding sequence for a gRNA under the control of aninducible promoter, and a selectable marker.

At step 8059, cells are grown until they reach stationary phase, ornearly so. Once the cells reach the stationary phase, editing is induced8067 (e.g., where transcription of the nuclease, gRNA or both isinduced) and the cells in the culture 8082 are edited and then allowedto recover from editing. Once recovered, the cells can be plated 8069,grown and pooled 8084. Alternatively, the cells from culture 8082 can beplated 8081, and slow-growing colonies are selected 8086 thereby cherrypicking edited colonies. In yet another alternative, the cells can beretained in liquid culture, grown to an appropriate OD, renderedelectrocompetent, and subjected to another round of editing 8088. Thismethod of enrichment of edited cells is particularly desirable as may beperformed in a high throughput manner and does not require plating cellsand is automatable. Induction at step 8067 can take place by, e.g.,using a pL promoter system where the pL promoter is induced by raisingthe temperature of the cells in the medium to 42° C. for, e.g., one tomany hours to induce expression of the nuclease and gRNA for cutting andediting. Once editing has been induced, the temperature of the culture8082 is returned to 30° C.

In one method 8081, the cells from the bulk liquid culture are platedand the slow-growing colonies are selected 8086. In edited cells, cellviability is compromised in the period after editing is induced. Theselection method shown in FIG. 8H (e.g., selecting slow growing colonies8081) takes advantage of the growth lag in colonies of edited cells toidentify edited cells. In some embodiments, the colony size of theedited cells is 20% smaller than colonies of non-edited cells. In someaspects the colony size of the edited cells is 30%, 40%, 50%, 60%, 70%,80% or 90% smaller than the colonies of non-edited cells. In manyembodiments, the colony size of the edited cells is 30-80% smaller thancolonies of non-edited cells, and in some embodiments, the colony sizeof the edited cells is 40-70% smaller than colonies of non-edited cells.

The Reagent Cartridge

FIG. 9A depicts a reagent cartridge 922 including a set of eighteentubes or vials 940. One or more of the tubes or vials 940, in someembodiments, is sealed with pierceable foil for access by an automatedliquid handling system, such as a sipper or pipettor. In otherembodiments, one or more of the tubes or vials may include a sealableaccess gasket. The top of each of the small tubes or vials, in someembodiments, is marked with machine-readable indicia (not illustrated)for automated identification of the contents. The machine-readableindicia may include a bar code, QR code, or other machine-readablecoding. Other automated means for identifying a particular container caninclude color coding, symbol recognition (e.g., text, image, icon,etc.), and/or shape recognition (e.g., a relative shape of thecontainer). Rather than being marked upon the vessel itself, in someembodiments, an upper surface of the cartridge body and/or the cartridgecover may contain machine-readable indicia for identifying contents. Thesmall tubes or vials may each be of a same size. Alternatively, multiplevolumes of tubes or vials may be provided in the reagent cartridge 922.In an illustrative example, each tube or vial may be designed to holdbetween 2 and 20 mL, between 4 and 10 mL, or about 5 mL. In someembodiments where only small volumes of some reagents are required, tubeinserts may be used to accommodate small (e.g., microfuge) tubes in alarger receptacle (not shown).

In an illustrative example, the tubes or vials may each hold one thefollowing materials: a vector backbone, oligonucleotides, reagents fornucleic acid assembly, a user-supplied cell sample, an inducer agent,magnetic beads in buffer, ethanol, an antibiotic for cell selection,reagents for eluting cells and nucleic acids, an oil overlay, otherreagents, and cell growth and/or recovery media. In addition, the celltransformation module such as the flow-through electroporation devicedescribed above optionally may be part of the reagent cartridge.

In some implementations, a cover 924 as seen in FIG. 9B secures thetubes or vials 940 within the cartridge body 922 of FIG. 9A. Turning toFIG. 9B, the cover 924 may include apertures for access to each of thesmall tubes or vials 940. Three large apertures 932 are outlined in abold band to indicate positions to add user-supplied materials. Theuser-supplied materials, for example, may include a vector backbone,oligonucleotides, and a cell sample. Further, the cover 924 may includemachine-readable indicia 930 for identifying the type of cartridge(e.g., accessing a map of the cartridge contents). Alternatively, eachaperture may be marked separately with the individual contents. In someimplementations, to ensure positioning of user-supplied materials, thevials or tubes provided for filling in the lab environment may haveunique shapes or sizes such that the cell sample vial or tube only fitsin the cell sample aperture, the oligonucleotides vial or tube only fitsin the oligonucleotides aperture, and so on.

Use of the Cell Growth Device

FIG. 10 is a flow chart of an example method 1000 for using an automatedmulti-module cell editing instrument such as the systems illustrated inFIGS. 4A-4D. A processing system, for example, directs the processingstage of the method 1000. For example, a software script may identifysettings for each processing stage and instructions for movement of arobotic handling system to perform the actions of the method 1000. Insome embodiments, a software instruction script may be identified by acartridge supplied to the automated multi-module cell editinginstrument. For example, the cartridge may include machine-readableindicia, such as a bar code or QR code, including identification of ascript stored in a memory of the automated multi-module cell editinginstrument. In another example, the cartridge may contain a downloadablescript embedded in machine-readable indicia such as a radio frequency(RF) tag. In other embodiments, the user may identify a script, forexample through downloading the script via a wired or wirelessconnection to the processing system of the automated multi-module cellediting instrument or through selecting a stored script through a userinterface of the automated multi-module cell editing instrument. In aparticular example, the automated multi-module cell editing instrumentmay include a touch screen interface for submitting user settings andactivating cell processing.

In some implementations, the method 1000 begins with transferring cellsto a cell growth module (1002). The growth module may be any growthmodule amendable to automation such as, for example, the cell growthmodule 550 described in relation to FIGS. 5B-5D. In a particularexample, the processing system may direct the robotic handling system totransfer cells to the growth module. In another example, the cells maybe transferred from one a reagent cartridge to the growth module by therobotic handling system. In some embodiments, the growth vial maycontain growth media and be supplied, e.g., as part of a kit. In otherembodiments, the growth vial may be filled with medium transferred,e.g., via the liquid handling device, from a reagent container.

In some embodiments, prior to transferring the cells (e.g., from thereagent cartridge or from a vial added to the instrument),machine-readable indicia may be scanned upon the vial or other containersituated in a position designated for cells to confirm that the vial orcontainer is marked as containing cells. Further, the machine-readableindicia may indicate a type of cells provided to the instrument. Thetype of cells, in some embodiments, may cause the instrument to select aparticular processing script (e.g., series of instructions for therobotic handling system and settings and activation of the variousmodules).

In some implementations, the cells are grown in the growth module to adesired optical density (1004). For example, the processing system maymanage a temperature setting of the growth module for incubating thecells during the growth cycle. The processing system may further receivesensor signals from the growth module indicative of optical density andanalyze the sensor signals to monitor growth of the cells. In someembodiments, a user may set growth parameters for managing growth of thecells. For example, temperature, and the degree of agitation of thecells. Further, in some embodiments, the user may be updated regardinggrowth process. The updates, in some examples, may include a messagepresented on a user interface of the automated multi-module cell editinginstrument, a text message to a user's cell phone number, an emailmessage to an email account, or a message transmitted to an appexecuting upon a portable electronic device (e.g., cell phone, tablet,etc.). Responsive to the messages, in some embodiments, the user maymodify parameters, such as temperature, to adjust cell growth. Forexample, the user may submit updated parameters through a user interfaceof the automated multi-module cell editing instrument or through aportable computing device application in communication with theautomated multi-module cell editing instrument, such as a user interface(see element 450 of FIG. 4B).

Although described in relation to optical density, in otherimplementations cell growth within the growth module may be monitoredusing a different measure of cell density and physiological state suchas, in some examples, pH, dissolved oxygen, released enzymes, acousticproperties, and electrical properties.

In some implementations, upon reaching the desired optical density(1004), the cells are transferred from the growth module to a filtrationmodule or cell wash and concentration module (1006). The robotichandling system, for example, may transfer the cells from the growthmodule to the cell concentration module. The cell concentration module,for example, may be (and typically is) designed to render the cellselectrocompetent. See FIG. 6A-6I in relation to the TFF device, above.The cells are rendered electrocompetent and eluted in the filtrationmodule or cell wash and concentration module (1008). The cells may beeluted using a wash solution. For example, the cells may be eluted usingreagents from a reagent supply.

Once the cells have been rendered electrocompetent and suspended in anappropriate volume such as 50 μL to 10 mL, or 100 μL to 80 mL, or 150 μLto 8 mL, or 250 μL to 7 mL, or 500 μL to 6 mL, or 750 μL to 5 mL fortransformation (1006), the cells are transferred to, e.g., an FTEPmodule (1018). The robotic handling system, for example, may transferthe cells from the filtration module to the FTEP. The filtration modulemay be physically coupled to the FTEP device, or these modules may beseparate.

In some implementations, nucleic acids are prepared outside of theautomated multi-module cell editing instrument. For example, anassembled vector or other nucleic acid assembly may be included as areagent by a user prior to running the transformation process and otherprocesses in the method 1000.

However, in other implementations, nucleic acids are prepared by theautomated multi-module cell editing instrument. A portion of thefollowing steps 1010 through 1016, in some embodiments, are performed inparallel with a portion of steps 1002 through 1008. At least a portionof the following steps, in some embodiments, are performed before and/orafter steps 1002 through 1008.

In some implementations, nucleic acids such as an editingoligonucleotide and a vector backbone, as well as in some examples,enzymes and other reaction components are transferred to a nucleic acidassembly module (1010). The nucleic acid assembly module may beconfigured to perform one or more of a wide variety of different nucleicacid assembly techniques in an automated fashion. Nucleic acid assemblytechniques that can be performed in the nucleic acid assembly module mayinclude, but are not limited to, those assembly methods that userestriction endonucleases, including PCR, BioBrick assembly, Type IIScloning, GoldenGate assembly, and Ligase Cycling Reaction. In otherexamples, the nucleic acid assembly module may perform an assemblytechnique based on overlaps between adjacent parts of the nucleic acids,such as Gibson Assembly®, CPEC, SLIC, Ligase Cycling, etc., as describedabove. Additional example assembly methods that may be performed by thenucleic acid assembly module include gap repair in yeast, gatewaycloning and topoisomerase-mediated cloning. In a particular example, theprocessing system may direct the robotic handling system to transfernucleic acids to the nucleic acid assembly module. In another example,the nucleic acids may be transferred from a reagent cartridge to anucleic acid assembly module by the robotic handling system.

In some embodiments—prior to transferring each of the nucleic acidsamples, the enzymes, and other reaction components—machine-readableindicia may be scanned upon the vials or other containers situated inpositions designated for these materials to confirm that the vials orcontainers are marked as containing the anticipated material. Further,the machine-readable indicia may indicate a type of one or more of thenucleic acid samples, the enzymes, and other reaction componentsprovided to the instrument. The type(s) of materials, in someembodiments, may cause the instrument to select a particular processingscript (e.g., series of instructions for the robotic handling system toidentify further materials and/or settings and activation of the nucleicacid assembly module).

In some embodiments, the nucleic acid assembly module is temperaturecontrolled depending upon the type of nucleic acid assembly used. Forexample, when PCR is utilized in the nucleic acid assembly module, themodule can have a thermocycling capability allowing the temperatures tocycle between denaturation, annealing and extension steps. When singletemperature assembly methods are utilized in the nucleic acid assemblymodule, the module can have the ability to reach and hold at thetemperature that optimizes the specific assembly process beingperformed.

Temperature control, in some embodiments, is managed by a processingsystem of the automated multi-module cell editing instrument, such asthe processing system. These temperatures and the duration ofmaintaining the temperatures can be determined by a preprogrammed set ofparameters (e.g., identified within the processing script or in anothermemory space accessible by the processing system), or manuallycontrolled by the user through interfacing with the processing system.

Once sufficient time has elapsed for the assembly reaction to takeplace, in some implementations, the nucleic acid assembly may betransferred to a purification module (1014). The processing system, forexample, may monitor timing of the assembly reaction based upon one ormore of the type of reaction, the type of materials, and user settingsprovided to the automated multi-module cell editing instrument. Therobotic handling system, for example, may transfer the nucleic acidassembly to the purification module through a sipper or pipettorinterface. In another example, the robotic handling system may transfera vial containing the nucleic acid assembly from a chamber of thenucleic acid assembly module to a chamber of the de-salt/purificationmodule.

In some implementations, the nucleic acid assembly is de-salted andeluted at the purification module (1016). The purification module, forexample, may remove unwanted components of the nucleic acid assemblymixture (e.g., salts, minerals, etc.). In some embodiments, thepurification module concentrates the assembled nucleic acids into asmaller volume that the nucleic acid assembly volume. Examples ofmethods for exchanging liquid following nucleic acid assembly includemagnetic beads (e.g., SPRI or Dynal (Dynabeads) by Invitrogen Corp. ofCarlsbad, Calif.), silica beads, silica spin columns, glass beads,precipitation (e.g., using ethanol or isopropanol), alkaline lysis,osmotic purification, extraction with butanol, membrane-based separationtechniques, filtration etc. For example, one or more micro-concentratorsfitted with anisotropic, hydrophilic-generated cellulose membranes ofvarying porosities may be used. In another example, thede-salt/purification module may process a liquid sample including anucleic acid and an ionic salt by contacting the mixture with an ionexchanger including an insoluble phosphate salt, removing the liquid,and eluting nucleic acid from the ion exchanger.

In an illustrative embodiment, the nucleic acid assembly may be combinedwith magnetic beads, such as SPRI beads, in a chamber of a purificationmodule. The nucleic acid assembly may be incubated at a set temperaturefor sufficient time for the assembled nucleic acids to bind to themagnetic beads. After incubation, a magnet may be engaged proximate tothe chamber so that the nucleic acid assembly can be washed and eluted.Once the nucleic acid assembly has been eluted, the nucleic acidassembly is transferred to the transformation module (1018). The robotichandling system, for example, may transfer the assembled nucleic acidsto the transformation module through a sipper or pipettor interface tothe FTEP as described above. For example, the de-salted assemblednucleic acids, during the transfer, may be combined with theelectrocompetent cells from step 108. In other embodiments, thetransformation module may accept each of the electrocompetent cells andthe nucleic acid assembly separately and enable the mixing (e.g., openone or more channels to combine the materials in a shared chamber).

The cells are transformed in the FTEP module (1020). A buffer or mediummay be transferred to the transformation module and added to the cellsso that the cells may be suspended in a buffer or medium that isfavorable for cell survival during electroporation. Prior totransferring the buffer or medium, machine-readable indicia may bescanned upon the vial or other container or reservoir situated in theposition designated for the buffer or medium to confirm the contents ofthe vial, container, or reservoir. Further, the machine-readable indiciamay indicate a type of buffer or medium provided to the instrument. Thetype of buffer or medium, in some embodiments, may cause the instrumentto select a particular processing script (e.g., settings and activationof the transformation module appropriate for the particular buffer ormedium). For bacterial cell electroporation, low conductance mediums,such as water or glycerol solutions, may be used to reduce the heatproduction by transient high current. For yeast cells a sorbitolsolution may be used. For mammalian cell electroporation, cells may besuspended in a highly conductive medium or buffer, such as MEM, DMEM,IMDM, RPMI, Hanks', PBS, HBSS, HeBS and Ringer's solution. In aparticular example, the robotic handling system may transfer a buffersolution to FTEP module from the reagent cartridge. As described inrelation to FIGS. 7A-7E, the FTEP device may be a disposable FTEP deviceand/or the FTEP device may be provided as part of the reagent cartridge.Alternatively, as shown in FIG. 4A, the FTEP device may a separatemodule.

Once transformed, the cells are transferred to a secondgrowth/recovery/editing module (1022) such as the cell growth moduledescribed in relation to FIGS. 5A-5D. The robotic handling system, forexample, may transfer the transformed cells to the second growth modulethrough a sipper or pipettor interface. In another example, the robotichandling system may transfer a vial containing the transformed cellsfrom a chamber of the transformation module to a chamber of the secondgrowth module.

The second growth module, in some embodiments, acts as a recoverymodule, allowing the cells to recover from the transformation process.In other embodiments, the cells may be provided to a separate recoverymodule prior to being transported to the second growth module. Duringrecovery, the second growth module allows the transformed cells touptake and, in certain aspects, integrate the introduced nucleic acidsinto the genome of the cell. The second growth module may be configuredto incubate the cells at any user-defined temperature optimal for cellgrowth, preferably 25°, 30°, or 37° C.

In some embodiments, the second growth module behaves as a selectionmodule, selecting the transformed cells based on an antibiotic or otherreagent. In one example, the RNA-guided nuclease (RGN) protein system isused for selection to cleave the genomes of cells that have not receivedthe desired edit. In the example of an antibiotic selection agent, theantibiotic may be added to the second growth module to enact selection.Suitable antibiotic resistance genes include, but are not limited to,genes such as ampicillin-resistance gene, tetracycline-resistance gene,kanamycin-resistance gene, neomycin-resistance gene,canavanine-resistance gene, blasticidin-resistance gene,hygromycin-resistance gene, puromycin-resistance gene, orchloramphenicol-resistance gene. The robotic handling system, forexample, may transfer the antibiotic to the second growth module througha sipper or pipettor interface. In some embodiments, removing dead cellbackground is aided using lytic enhancers such as detergents, osmoticstress by hyponic wash, temperature, enzymes, proteases, bacteriophage,reducing agents, or chaotropes. The processing system, for example, mayalter environmental variables, such as temperature, to induce selection,while the robotic handling system may deliver additional materials(e.g., detergents, enzymes, reducing agents, etc.) to aid in selection.In other embodiments, cell removal and/or media exchange by filtrationis used to reduce dead cell background.

In further embodiments, in addition to or as an alternative to applyingselection, the second growth module serves as an editing module,allowing for genome editing in the transformed cells. Alternatively, inother embodiments the cells post-recovery and selection (if performed)are transferred to a separate editing module. As an editing module, thesecond growth module induces editing of the cells' genomes, e.g.,through facilitating expression of the introduced nucleic acids.Expression of the nuclease and/or editing cassette nucleic acids mayinvolve one or more of chemical, light, viral, or temperature inductionmethods. The second growth module, for example, may be configured toheat or cool the cells during a temperature induction process. In aparticular illustration, the cells may be induced by heating at 42°C.-50° C. Further to the illustration, the cells may then be are cooledto 0-10° C. after induction. In the example of chemical or viralinduction, an inducing agent may be transferred to the second growthmodule to induce editing. If an inducible nuclease and/or editingcassette was introduced to the cells during editing, it can be inducedthrough introduction of an inducer molecule. The inducing agent orinducer molecule, in some implementations, is transferred to the secondgrowth module by the robotic handling system, e.g., through a pipettoror sipper interface.

In some implementations, if no additional cell editing is desired(1024), the cells may be transferred from the cell growth module to astorage unit for later removal from the automated multi-module cellediting instrument (1026). The robotic handling system, for example, maytransfer the cells to a storage unit through a sipper or pipettorinterface. In another example, the robotic handling system may transfera vial containing the cells from a chamber of the second growth moduleto a vial or tube within the storage unit.

In some implementations, if additional cell editing is desired (1024),the cells may be transferred to the same or a different filtrationmodule and rendered electrocompetent (1008). Further, in someembodiments, a new assembled nucleic acid sample may be prepared by thenucleic acid assembly module at this time, or, alternatively, a secondfully assembled nucleic acid may be directly introduced to the cells.Prior to recursive editing, in some embodiments, the automatedmulti-module cell editing instrument may require additional materials besupplied by the user, e.g., through the introduction of one or moreseparate reagents vails or cartridge.

The steps may be the same or different during the second round ofediting. For example, in some embodiments, upon a subsequent executionof step 1004, a selective growth medium is transferred to the growthmodule to enable selection of edited cells from the first round ofediting. The robotic handling system may transfer the selective growthmedium from a vial or container in a reagent cartridge situated in aposition designated for selective growth medium. Prior to transferringthe selective growth medium, machine-readable indicia may be scannedupon the vial or other container or reservoir situated in the positiondesignated for the selective growth medium to confirm the contents ofthe vial, container, or reservoir. Further, the machine-readable indiciamay indicate a type of selective growth medium provided to theinstrument. The type of selective growth medium, in some embodiments,may cause the instrument to select a particular processing script (e.g.,settings and activation of the growth module appropriate for theparticular selective growth medium). Particular examples of recursiveediting workflows are described in relation to FIG. 13.

In some implementations, the method 1000 can be timed to introducematerials and/or complete the editing cycle or growth cycle incoordination with a user's schedule. For example, the automatedmulti-module cell editing instrument may provide the user the ability toschedule completion of one or more cell processing cycles (e.g., one ormore recursive edits) such that the method 1000 is enacted with a goalof completion at the user's preferred time. The time scheduling, forexample, may be set through a user interface. For illustration only, auser may set completion of a first cycle to 4:00 PM so that the user cansupply additional cartridges of materials to the automated multi-modulecell editing instrument to enable overnight processing of another roundof cell editing. Thus a user may time the programs so that two or morecycles may be programmed in a specific time period, e.g., a 24-hourperiod.

In some implementations, throughout the method 1000, the automatedmulti-module cell editing instrument may alert the user to its currentstatus. For example, the user interface may present a graphicalindication of the present stage of processing. In a particular example,a front face of the automated multi-module call processing instrumentmay be overlaid with a user interface (e.g., touch screen) that presentsan animated graphic depicting present status of the cell processing. Theuser interface may further present any user and/or default settingsassociated with the current processing stage (e.g., temperature setting,time setting, etc.). In certain implementations, the status may becommunicated to a user via wireless communications controller.

Although illustrated as a particular series of operations, in otherembodiments, more or fewer steps may be included in the method 1000. Forexample, in some embodiments, prior to engaging in each round ofediting, the contents of reservoirs, cartridges, and/or vials may bescreened to confirm appropriate materials are available to proceed withprocessing. For example, in some embodiments, one or more imagingsensors (e.g., barcode scanners, cameras, etc.) may confirm contents atvarious locations within the housing of the automated multi-module cellediting instrument. In one example, multiple imaging sensors may bedisposed within the housing of the automated multi-module cell editinginstrument, each imaging sensor configured to detect one or morematerials (e.g., machine-readable indicia such as barcodes or QR codes,shapes/sizes of materials, etc.). In another example, at least oneimaging sensor may be moved by the robotic handling system to multiplelocations to detect one or more materials. In further embodiments, oneor more weight sensors may detect presence or absence of disposable orreplaceable materials. In an illustrative example, the transfer tipsupply holder may include a weight sensor to detect whether or not tipshave been loaded into the region. In another illustrative example, anoptical sensor may detect that a level of liquid waste has reached athreshold level, requiring disposal prior to continuation of cellprocessing or addition of liquid if the minimum level has not beenreached to proceed. Requests for additional materials, removal of wastesupplies, or other user interventions (e.g., manual cleaning of one ormore elements, etc.), in some implementations, are presented on agraphical user interface of the automated multi-module cell editinginstrument. The automated multi-module cell editing instrument, in someimplementations, contacts the user with requests for new materials orother manual interventions, for example through a software app, email,or text message.

FIG. 11 shows simplified flow charts for two alternative exemplarymethods 1100 a and 1100 b for singulating cells for enrichment (1100 a)and for cherry picking (1100 b). Looking at FIG. 11, method 1100 abegins by transforming cells 1110 with the components necessary toperform nucleic acid-guided nuclease editing. For example, the cells maybe transformed simultaneously with separate engine and editing vectors;the cells may already be expressing the nuclease (e.g., the cells mayhave already been transformed with an engine vector or the codingsequence for the nuclease may be stably integrated into the cellulargenome) such that only the editing vector needs to be transformed intothe cells; or the cells may be transformed with a single vectorcomprising all components required to perform nucleic acid-guidednuclease genome editing.

As described above, a variety of delivery systems can be used tointroduce (e.g., transform or transfect) nucleic acid-guided nucleaseediting system components into a host cell 1110. These delivery systemsinclude the use of yeast systems, lipofection systems, microinjectionsystems, biolistic systems, virosomes, liposomes, immunoliposomes,polycations, lipid:nucleic acid conjugates, virions, artificial virions,viral vectors, electroporation, cell permeable peptides, nanoparticles,nanowires, exosomes. Alternatively, molecular trojan horse liposomes maybe used to deliver nucleic acid-guided nuclease components across theblood brain barrier. Of interest, particularly in the context of amulti-module cell editing instrument is the use of electroporation,particularly flow-through electroporation (either as a stand-aloneinstrument or as a module in an automated multi-module system) asdescribed in, e.g., U.S. Ser. No. Ser. No. 16/147,120, filed 28 Sep.2018; Ser. No. 16/147,353, filed 28 Sep. 2018; Ser. No. 16/147,865,filed 30 Sep. 2018; and Ser. No. 16/147,871, filed 30 Sep. 2018. If thesolid wall singulation/growth/editing/normalization module is one modulein an automated multi-module cell editing instrument, the cells arelikely transformed in an automated cell transformation module.

After the cells are transformed with the components necessary to performnucleic acid-guided nuclease editing, the cells are singulated inmicrowells in a, e.g., solid wall device 1120; that is, the cells arediluted (if necessary) in a liquid culture medium (in some embodiments,including Tween, at a concentration of 0.1% or less to effect a gooddistribution) so that the cells, when delivered to the solid walldevice, fill the microwells of the solid wall device in a Poisson orsubstantial Poisson distribution. Singulation is accomplished when anaverage of ½ cell is delivered to each microwell; that is, where somemicrowells contain one cell and other microwells contain no cells.

Once the cells in this embodiment have been singulated in 1100 a, thecells are actively editing, as the editing “machinery” is under thecontrol of a constitutive promoter. As the cells are editing, they aregrown into colonies of terminal size 1130; that is, the colonies arisingfrom the singulated cells are grown into colonies to a point where cellgrowth has peaked and is normalized or saturated for both edited andunedited cells. Normalization occurs as the nutrients in the mediumaround a growing cell colony are depleted and/or cell growth fills themicrowells and further growth is constrained. Again, in the embodiment1100 a shown in FIG. 11, the editing components are under the control ofa constitutive promoter; thus, editing begins immediately (or almostimmediately) upon transformation. However, in other embodiments such asthe embodiment shown in 1100 b described below, one or both of thenuclease and the guide nucleic acid (as well as, e.g., the λ redrecombination system components in bacterial systems) may be under thecontrol of an inducible promoter, in which case editing may be inducedafter, e.g., a desired number of cell doublings. Turning back to method1100 a, the terminal-size colonies are pooled 1140 by flushing theclonal cell colonies from the microwells to mix the cells from thenormalized cell colonies. Again, because singulation overcomes growthbias from unedited cells or cells exhibiting fitness effects as theresult of edits made, singulation/normalization alone enriches the totalpopulation of cells with cells that have been edited; that is,singulation combined with normalization (e.g., growing colonies toterminal size) allows for high-throughput enrichment of edited cells.

The method 1100 b shown in FIG. 11 is similar to the method 1100 a inthat cells of interest are transformed 1110 with the componentsnecessary to perform nucleic acid-guided nuclease editing. As describedabove, the cells may be transformed simultaneously with both the engineand editing vectors, the cells may already be expressing the nuclease(e.g., the cells may have already been transformed with an engine vectoror the coding sequence for the nuclease may be stably integrated intothe cellular genome) such that only the editing vector needs to betransformed into the cells, or the cells may be transformed with asingle vector comprising all components required to perform nucleicacid-guided nuclease genome editing. Further, if thesingulation/growth/editing/normalization solid wall module is one modulein an automated multi-module cell editing instrument, celltransformation may be performed in an automated transformation module asdescribed above.

After the cells are transformed with the components necessary to performnucleic acid-guided nuclease editing, the cells are diluted (ifnecessary) in liquid medium so that the cells, when delivered to thesolid wall device, fill the microwells of the solid wall device in aPoisson or substantial Poisson distribution.

Once the cells have been singulated in the microwells of the solid walldevice 1120, the cells are allowed to grow to, e.g., between 2 and 150,or between 5 and 120, or between 10 and 100 doublings, establishingclonal colonies 1150. After colonies are established, in this embodiment1100 b editing is induced 1160 by, e.g., activating inducible promotersthat control transcription of one or more of the components needed fornucleic acid-guided nuclease editing, such as, e.g., transcription ofthe gRNA, nuclease, or, in the case of bacteria, a recombineeringsystem. Once editing is induced 1160, many of the edited cells in theclonal colonies die due to the double-strand DNA breaks that occurduring the editing process; however, in a percentage of edited cells,the genome is edited and the double strand break is properly repaired.These edited cells then start growing and re-establish colonies;however, the growth of edited colonies tends to lag behind the growth ofclonal colonies where an edit has not taken place. The small orslow-growing colonies (edited cells) are cherry picked 1170.

FIG. 12 is a simplified block diagram of an embodiment of an exemplaryautomated multi-module cell processing instrument comprising a solidwall singulation/growth/editing/normalization module for enrichment foredited cells. The cell processing instrument 1200 may include a housing1244, a reservoir of cells to be transformed or transfected 1202, and agrowth module (a cell growth device) 1204. The cells to be transformedare transferred from a reservoir to the growth module to be cultureduntil the cells hit a target OD. Once the cells hit the target OD, thegrowth module may cool or freeze the cells for later processing, or thecells may be transferred to a filtration module 1230 where the cells arerendered electrocompetent and concentrated to a volume optimal for celltransformation. Once concentrated, the cells are then transferred to theelectroporation device 1208 (e.g., transformation/transfection module).Exemplary electroporation devices of use in the automated multi-modulecell processing instruments for use in the multi-module cell processinginstrument include flow-through electroporation devices such as thosedescribed in U.S. Ser. No. 16/147,120, filed 28 Sep. 2018; Ser. No.16/147,353, filed 28 Sep. 2018; Ser. No. 16/147,865, filed 30 Sep. 2018;and Ser. No. 16/147,871, filed 30 Sep. 2018 all of which are hereinincorporated by reference in their entirety.

In addition to the reservoir for storing the cells, the system 1200 mayinclude a reservoir for storing editing oligonucleotide cassettes 1216and a reservoir for storing an expression vector backbone 1218. Both theediting oligonucleotide cassettes and the expression vector backbone aretransferred from the reagent cartridge to a nucleic acid assembly module1220, where the editing oligonucleotide cassettes are inserted into theexpression vector backbone. The assembled nucleic acids may betransferred into an optional purification module 1222 for desaltingand/or other purification and/or concentration procedures needed toprepare the assembled nucleic acids for transformation. Alternatively,pre-assembled nucleic acids, e.g., an editing vector, may be storedwithin reservoir 1216 or 1218. Once the processes carried out by thepurification module 1222 are complete, the assembled nucleic acids aretransferred to, e.g., an electroporation device 1205, which alreadycontains the cell culture grown to a target OD and renderedelectrocompetent via filtration module 1230. In electroporation device1208, the assembled nucleic acids are introduced into the cells.Following electroporation, the cells are transferred into a combinedrecovery/selection module 1210.

Following recovery, and, optionally, selection, the cells aretransferred to a singulation, editing, and growth module 1240, where thecells are diluted and compartmentalized such that there is an average ofone cell per compartment. Once singulated, the cells are allowed to growfor a pre-determined number of doublings. Once these initial coloniesare established, editing is induced and the edited cells are allowed toestablish colonies, which are grown to terminal size (e.g., the coloniesare normalized). In some embodiments, editing is induced by one or moreof the editing components being under the control of an induciblepromoter. In some embodiments, the inducible promoter is activated by arise in temperature and “deactivated” by lowering the temperature.Alternatively, in embodiments where the singulation device is a solidwall device comprising a filter forming the bottom of the microwell, thesolid wall device can be transferred to a plate (e.g., agar plate oreven to liquid medium) comprising a medium with a component thatactivates or induced editing, then transferred to a medium thatdeactivates editing. Once the colonies are grown to terminal size, thecolonies are pooled. Again, singulation overcomes growth bias fromunedited cells and growth bias resulting from fitness effects ofdifferent edits.

The recovery, selection, singulation, induction, editing and growthmodules may all be separate, may be arranged and combined as shown inFIG. 12, or may be arranged or combined in other configurations. Incertain embodiments, all of recovery, selection, singulation, growth,editing, and normalization are performed in a solid wall device.Alternatively, recovery, selection, and dilution, if necessary, areperformed in liquid medium in a separate vessel (module), thentransferred to the solid wallsingulation/growth/induction/editing/normalization module.

Once the normalized cell colonies are pooled, the cells may be stored,e.g., in a storage module 1212, where the cells can be kept at, e.g., 4°C. until the cells are retrieved for further study. Alternatively, thecells may be used in another round of editing. The multi-module cellprocessing instrument is controlled by a processor 1242 configured tooperate the instrument based on user input, as directed by one or morescripts, or as a combination of user input or a script. The processor1242 may control the timing, duration, temperature, and operations ofthe various modules of the system 500 and the dispensing of reagents.For example, the processor 1242 may cool the cells post-transformationuntil editing is desired, upon which time the temperature may be raisedto a temperature conducive of genome editing and cell growth. Theprocessor may be programmed with standard protocol parameters from whicha user may select, a user may specify one or more parameters manually orone or more scripts associated with the reagent cartridge may specifyone or more operations and/or reaction parameters. In addition, theprocessor may notify the user (e.g., via an application to a smart phoneor other device) that the cells have reached the target OD as well asupdate the user as to the progress of the cells in the various modulesin the multi-module system.

The automated multi-module cell processing instrument 1200 is anuclease-directed genome editing system and can be used in singleediting systems (e.g., introducing one or more edits to a cellulargenome in a single editing process). The system of FIG. 13, describedbelow, is configured to perform sequential editing, e.g., usingdifferent nuclease-directed systems sequentially to provide two or moregenome edits in a cell; and/or recursive editing, e.g. utilizing asingle nuclease-directed system to introduce sequentially two or moregenome edits in a cell.

FIG. 13 illustrates another embodiment of a multi-module cell processinginstrument. This embodiment depicts an exemplary system that performsrecursive gene editing on a cell population. As with the embodimentshown in FIG. 12, the cell processing instrument 1300 may include ahousing 1344, a reservoir for storing cells to be transformed ortransfected 1302, and a cell growth module (comprising, e.g., a rotatinggrowth vial) 1304. The cells to be transformed are transferred from areservoir to the cell growth module to be cultured until the cells hit atarget OD. Once the cells hit the target OD, the growth module may coolor freeze the cells for later processing or transfer the cells to afiltration module 1360 where the cells are subjected to buffer exchangeand rendered electrocompetent, and the volume of the cells may bereduced substantially. Once the cells have been concentrated to anappropriate volume, the cells are transferred to electroporation device1308. In addition to the reservoir for storing cells, the multi-modulecell processing instrument includes a reservoir for storing the vectorpre-assembled with editing oligonucleotide cassettes 1352. Thepre-assembled nucleic acid vectors are transferred to theelectroporation device 1308, which already contains the cell culturegrown to a target OD. In the electroporation device 1308, the nucleicacids are electroporated into the cells. Following electroporation, thecells are transferred into an optional recovery module 1356, where thecells are allowed to recover briefly post-transformation.

After recovery, the cells may be transferred to a storage module 1312,where the cells can be stored at, e.g., 4° C. for later processing, orthe cells may be diluted and transferred to aselection/singulation/growth/induction/editing/normalization module1358. In the singulation/edit/growth module 1358, the cells are arrayedsuch that there is an average of one cell per microwell. The arrayedcells may be in selection medium to select for cells that have beentransformed or transfected with the editing vector(s). Once singulated,the cells grow through 2-50 doublings and establish colonies. Oncecolonies are established, editing is induced by providing conditions(e.g., temperature, addition of an inducing or repressing chemical) toinduce editing. Once editing is initiated and allowed to proceed, thecells are allowed to grow to terminal size (e.g., normalization of thecolonies) in the microwells and then can be flushed out of themicrowells and pooled, then transferred to the storage (or recovery)unit 1314 or can be transferred to a growth module 1304 for anotherround of editing. In between pooling and transfer to a growth module,there may be one or more additional steps, such as cell recovery, mediumexchange, cells concentration, etc., by, e.g., filtration. Note that theselection/singulation/growth/induction/editing and normalization modulesmay be the same module, where all processes are performed in the solidwall device, or selection and/or dilution may take place in a separatevessel before the cells are transferred to the solid wallsingulation/growth/induction/editing/normalization module (solid walldevice). As an alternative to singulation in, e.g., a solid wall device,the transformed cells may be grown in—and editing can be induced in—bulkliquid as described above in relation to FIGS. 8F-8H above. Once theputatively-edited cells are pooled, they may be subjected to anotherround of editing, beginning with growth, cell concentration andtreatment to render electrocompetent, and transformation by yet anotherdonor nucleic acid in another editing cassette via the electroporationmodule 1308.

In electroporation device 1308, the cells selected from the first roundof editing are transformed by a second set of editing oligos (or othertype of oligos) and the cycle is repeated until the cells have beentransformed and edited by a desired number of, e.g., editing cassettes.The multi-module cell processing instrument exemplified in FIG. 13 iscontrolled by a processor 1342 configured to operate the instrumentbased on user input or is controlled by one or more scripts including atleast one script associated with the reagent cartridge. The processor1342 may control the timing, duration, and temperature of variousprocesses, the dispensing of reagents, and other operations of thevarious modules of the instrument 1300. For example, a script or theprocessor may control the dispensing of cells, reagents, vectors, andediting oligonucleotides; which editing oligonucleotides are used forcell editing and in what order; the time, temperature and otherconditions used in the recovery and expression module, the wavelength atwhich OD is read in the cell growth module, the target OD to which thecells are grown, and the target time at which the cells will reach thetarget OD. In addition, the processor may be programmed to notify a user(e.g., via an application) as to the progress of the cells in theautomated multi-module cell processing instrument.

It should be apparent to one of ordinary skill in the art given thepresent disclosure that the process described may be recursive andmultiplexed; that is, cells may go through the workflow described inrelation to FIG. 13, then the resulting edited culture may go throughanother (or several or many) rounds of additional editing (e.g.,recursive editing) with different editing vectors. For example, thecells from round 1 of editing may be diluted and an aliquot of theedited cells edited by editing vector A may be combined with editingvector B, an aliquot of the edited cells edited by editing vector A maybe combined with editing vector C, an aliquot of the edited cells editedby editing vector A may be combined with editing vector D, and so on fora second round of editing. After round two, an aliquot of each of thedouble-edited cells may be subjected to a third round of editing, where,e.g., aliquots of each of the AB-, AC-, AD-edited cells are combinedwith additional editing vectors, such as editing vectors X, Y, and Z.That is that double-edited cells AB may be combined with and edited byvectors X, Y, and Z to produce triple-edited edited cells ABX, ABY, andABZ; double-edited cells AC may be combined with and edited by vectorsX, Y, and Z to produce triple-edited cells ACX, ACY, and ACZ; anddouble-edited cells AD may be combined with and edited by vectors X, Y,and Z to produce triple-edited cells ADX, ADY, and ADZ, and so on. Inthis process, many permutations and combinations of edits can beexecuted, leading to very diverse cell populations and cell libraries.In any recursive process, it is advantageous to “cure” the previousengine and editing vectors (or single engine+editing vector in a singlevector system). “Curing” is a process in which one or more vectors usedin the prior round of editing is eliminated from the transformed cells.Curing can be accomplished by, e.g., cleaving the vector(s) using acuring plasmid thereby rendering the editing and/or engine vector (orsingle, combined vector) nonfunctional; diluting the vector(s) in thecell population via cell growth (that is, the more growth cycles thecells go through, the fewer daughter cells will retain the editing orengine vector(s)), or by, e.g., utilizing a heat-sensitive origin ofreplication on the editing or engine vector (or combined engine+editingvector). The conditions for curing will depend on the mechanism used forcuring; that is, in this example, how the curing plasmid cleaves theediting and/or engine plasmid.

FIG. 14 is a simplified block diagram of an embodiment of an exemplaryautomated multi-module cell processing instrument comprising a bulkliquid growth module for induced editing and enrichment for edited cellsas described above in relation to FIGS. 8H-8F. The cell processinginstrument 1400 may include a housing 1444, a reservoir of cells to betransformed or transfected 1402, and a growth module (a cell growthdevice) 1404. The cells to be transformed are transferred from areservoir to the growth module to be cultured until the cells hit atarget OD. Once the cells hit the target OD, the growth module may coolor freeze the cells for later processing, or the cells may betransferred to a filtration module 1430 where the cells are renderedelectrocompetent and concentrated to a volume optimal for celltransformation. Once concentrated, the cells are then transferred to anelectroporation device 1408 (e.g., transformation/transfection module).Exemplary electroporation devices of use in the automated multi-modulecell processing instruments for use in the multi-module cell processinginstrument include flow-through electroporation devices such as thosedescribed in U.S. Ser. No. 16/147,120, filed 28 Sep. 2018; Ser. No.16/147,353, filed 28 Sep. 2018; Ser. No. 16/147,865, filed 30 Sep. 2018;and Ser. No. 16/147,871, filed 30 Sep. 2018 all of which are hereinincorporated by reference in their entirety.

In addition to the reservoir for storing the cells, the system 1400 mayinclude a reservoir for storing editing cassettes 1416 and a reservoirfor storing an expression vector backbone 1418. Both the editingoligonucleotide cassettes and the expression vector backbone aretransferred from the reagent cartridge to a nucleic acid assembly module1420, where the editing oligonucleotide cassettes are inserted into theexpression vector backbone. The assembled nucleic acids may betransferred into an optional purification module 1422 for desaltingand/or other purification and/or concentration procedures needed toprepare the assembled nucleic acids for transformation. Alternatively,pre-assembled nucleic acids, e.g., an editing vector, may be storedwithin reservoir 1416 or 1418. Once the processes carried out by thepurification module 1422 are complete, the assembled nucleic acids aretransferred to, e.g., an electroporation device 1408, which alreadycontains the cell culture grown to a target OD and renderedelectrocompetent via filtration module 1430. In electroporation device1408, the assembled nucleic acids are introduced into the cells.Following electroporation, the cells are transferred into a combinedrecovery/selection module 1410. For examples of multi-module cellediting instruments, see U.S. Ser. Nos. 16/024,816 and 16/024,831, filed30 Jun. 2018, both of which are herein incorporated by reference intheir entirety.

Following recovery, and, optionally, selection, the cells aretransferred to a growth, induction, and editing module (bulk liquidculture) 1440. The cells are allowed to grow until the cells reach thestationary growth phase (or nearly so), then editing is induced byinduction of transcription of one or both of the nuclease and gRNA. Insome embodiments, editing is induced by transcription of one or both ofthe nuclease and the gRNA being under the control of an induciblepromoter. In some embodiments, the inducible promoter is a pL promoterwhere the promoter is activated by a rise in temperature and“deactivated” by lowering the temperature.

The recovery, selection, growth, induction, editing and storage modulesmay all be separate, may be arranged and combined as shown in FIG. 14,or may be arranged or combined in other configurations. In certainembodiments, recovery and selection are performed in one module, andgrowth, editing, and re-growth are performed in a separate module.Alternatively, recovery, selection, growth, editing, and re-growth areperformed in a single module.

Once the cells are edited and re-grown (e.g., recovered from editing),the cells may be stored, e.g., in a storage module 1412, where the cellscan be kept at, e.g., 4° C. until the cells are retrieved for furtherstudy. Alternatively, the cells may be used in another round of editing.The multi-module cell processing instrument is controlled by a processor1442 configured to operate the instrument based on user input, asdirected by one or more scripts, or as a combination of user input or ascript. The processor 1442 may control the timing, duration,temperature, and operations of the various modules of the system 1400and the dispensing of reagents. For example, the processor 1442 may coolthe cells post-transformation until editing is desired, upon which timethe temperature may be raised to a temperature conducive of genomeediting and cell growth. The processor may be programmed with standardprotocol parameters from which a user may select, a user may specify oneor more parameters manually or one or more scripts associated with thereagent cartridge may specify one or more operations and/or reactionparameters. In addition, the processor may notify the user (e.g., via anapplication to a smart phone or other device) that the cells havereached the target OD as well as update the user as to the progress ofthe cells in the various modules in the multi-module system.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Example 1: Growth in the Cell Growth Module

One embodiment of the cell growth device as described herein was testedagainst a conventional cell shaker shaking a 5 ml tube and an orbitalshaker shaking a 125 ml baffled flask to evaluate cell growth inbacterial and yeast cells. Additionally, growth of a bacterial cellculture and a yeast cell culture was monitored in real time using anembodiment of the cell growth device described herein.

In a first example, 20 ml EC23 cells (E. coli cells) in LB were grown ina 35 ml rotating growth vial with a 2-paddle configuration at 30° C.using the cell growth device as described herein. The rotating growthvial was spun at 600 rpm and oscillated (i.e., the rotation directionwas changed) every 1 second. In parallel, 5 ml EC23 cells in LB weregrown in a 5 ml tube at 30° C. and were shaken at 750 rpm. OD₆₀₀ wasmeasured at intervals using a NanoDrop™ spectrophotometer (Thermo FisherScientific). The results are shown in FIG. 15. The rotating growthvial/cell growth device performed better than the cell shaker in growingthe cells to OD₆₀₀ 2.6 in slightly over 4 hours. Another experiment wasperformed with the same conditions (volumes, cells, oscillation) theonly difference being a 3-paddle rotating growth vial was employed withthe cell growth device, and the results are shown in FIG. 16. Again, therotating growth vial/cell growth device performed better than the cellshaker in growing the cells to OD₆₀₀ 1.9.

Two additional experiments were performed, this time comparing therotating growth vial/cell growth device to a baffled flask and anorbital shaker. In one experiment, 20 ml EC138 cells (E. coli cells) inLB were grown in a 35 ml rotating growth vial with a 4-paddleconfiguration at 30° C. The rotating growth vial was spun at 600 rpm andoscillated (i.e., the rotation direction was changed) every 1 second. Inparallel, 20 ml EC138 cells in LB were grown in a 125 ml baffled flaskat 30° C. using an orbital shaker. OD₆₀₀ was measured at intervals usinga NanoDrop™ spectrophotometer (Thermo Fisher Scientific). The resultsare shown in FIG. 17, demonstrating that the rotating growth vial/cellgrowth device performed as well as the orbital shaker in growing thecells to OD₆₀₀ 1.0. In a second experiment 20 ml EC138 cells (E. colicells) in LB were grown in a 35 ml rotating growth vial with a 2-paddleconfiguration at 30° C. using the cell growth device as describedherein. The rotating growth vial was spun at 600 rpm and oscillated(i.e., the rotation direction was changed) every 1 second. In parallel,20 ml EC138 cells in LB were grown in a 125 ml baffled flask at 30° C.using an orbital shaker. OD₆₀₀ was measured at intervals using aNanoDrop™ spectrophotometer (Thermo Fisher Scientific). The results areshown in FIG. 18, demonstrating that the rotating growth vial/cellgrowth device performed as well—or better—as the orbital shaker ingrowing the cells to OD₆₀₀ 1.2.

In yet another experiment, the rotating growth vial/cell growth devicewas used to measure OD₆₀₀ in real time. FIG. 19 is a graph showing theresults of real time measurement of growth of an EC138 cell culture at30° C. using oscillating rotation and employing a 2-paddle rotatinggrowth vial. Note that OD₆₀₀ 2.6 was reached in 4.4 hours.

In another experiment, the rotating growth vial/cell growth device wasused to measure OD₆₀₀ in real time of yeast s288c cells in YPAD. Thecells were grown at 30° C. using oscillating rotation and employing a2-paddle rotating growth vial. FIG. 20 is a graph showing the results.Note that OD₆₀₀ 6.0 was reached in 14 hours.

Example 2: Cell Concentration

The TFF module as described above in relation to FIGS. 6A-6I has beenused successfully to process and perform buffer exchange on both E. coliand yeast cultures. In concentrating an E. coli culture, the followingsteps were performed:

First, a 20 ml culture of E. coli in LB grown to OD 0.5-0.62 was passedthrough the TFF device in one direction, then passed through the TFFdevice in the opposite direction. At this point the cells wereconcentrated to a volume of approximately 5 ml. Next, 50 ml of 10%glycerol was added to the concentrated cells, and the cells were passedthrough the TFF device in one direction, in the opposite direction, andback in the first direction for a total of three passes. Again the cellswere concentrated to a volume of approximately 5 ml. Again, 50 ml of 10%glycerol was added to the 5 ml of cells and the cells were passedthrough the TFF device for three passes. This process was repeated; thatis, again 50 ml 10% glycerol was added to cells concentrated to 5 ml,and the cells were passed three times through the TFF device. At the endof the third pass of the three 50 ml 10% glycerol washes, the cells wereagain concentrated to approximately 5 ml of 10% glycerol. The cells werethen passed in alternating directions through the TFF device three moretimes, wherein the cells were concentrated into a volume ofapproximately 400 μl.

Filtrate conductivity and filter processing time was measured for E.coli with the results shown in FIG. 21A. Filter performance wasquantified by measuring the time and number of filter passes required toobtain a target solution electrical conductivity. Cell retention wasdetermined by comparing the optical density (OD600) of the cell cultureboth before and after filtration. Filter health was monitored bymeasuring the transmembrane flow rate during each filter pass. Targetconductivity (˜16 μS/cm) was achieved in approximately 30 minutesutilizing three 50 ml 10% glycerol washes and three passes of the cellsthrough the device for each wash. The volume of the cells was reducedfrom 20 ml to 400 μl, and recovery of approximately 90% of the cells hasbeen achieved.

The same process was repeated with yeast cell cultures. A yeast culturewas initially concentrated to approximately 5 ml using two passesthrough the TFF device in opposite directions. The cells were washedwith 50 ml of 1M sorbitol three times, with three passes through the TFFdevice after each wash. After the third pass of the cells following thelast wash with 1M sorbitol, the cells were passed through the TFF devicetwo times, wherein the yeast cell culture was concentrated toapproximately 525 μl. FIG. 21B presents the filter buffer exchangeperformance for yeast cells determined by measuring filtrateconductivity and filter processing time. Target conductivity (˜10 μS/cm)was achieved in approximately 23 minutes utilizing three 50 ml 1Msorbitol washes and three passes through the TFF device for each wash.The volume of the cells was reduced from 20 ml to 525 μl. Recovery ofapproximately 90% of the cells has been achieved.

Example 3: Production and Transformation of Electrocompetent E. coli andS. Cerevisiae

For testing transformation of the FTEP device, electrocompetent E. colicells were created. To create a starter culture, 6 ml volumes of LBchlor-25 (LB with 25 μg/ml chloramphenicol) were transferred to 14 mlculture tubes. A 25 μl aliquot of E. coli was used to inoculate the LBchlor-25 tubes. Following inoculation, the tubes were placed at a 45°angle in the shaking incubator set to 250 RPM and 30° C. for overnightgrowth, between 12-16 hrs. The OD600 value should be between 2.0 and4.0. A 1:100 inoculum volume of the 250 ml LB chlor-25 tubes weretransferred to four sterile 500 ml baffled shake flasks, i.e., 2.5 mlper 250 ml volume shake flask. The flasks were placed in a shakingincubator set to 250 RPM and 30° C. The growth was monitored bymeasuring OD₆₀₀ every 1 to 2 hr. When the OD₆₀₀ of the culture wasbetween 0.5-0.6 (approx. 3-4 hrs), the flasks were removed from theincubator. The cells were centrifuged at 4300 RPM, 10 min, 4° C. Thesupernatant was removed, and 100 ml of ice-cold 10% glycerol wastransferred to each sample. The cells were gently resuspended, and thewash procedure performed three times, each time with the cellsresuspended in 10% glycerol. After the fourth centrifugation, the cellresuspension was transferred to a 50 ml conical Falcon tube andadditional ice-cold 10% glycerol added to bring the volume up to 30 ml.The cells were again centrifuged at 4300 RPM, 10 min, 4° C., thesupernatant removed, and the cell pellet resuspended in 10 ml ice-coldglycerol. The cells are aliquoted in 1:100 dilutions of cell suspensionand ice-cold glycerol.

The comparative electroporation experiment was performed to determinethe efficiency of transformation of the electrocompetent E. coli usingthe FTEP device described. The flow rate was controlled with a pressurecontrol system. The suspension of cells with DNA was loaded into theFTEP inlet reservoir. The transformed cells flowed directly from theinlet and inlet channel, through the flow channel, through the outletchannel, and into the outlet containing recovery medium. The cells weretransferred into a tube containing additional recovery medium, placed inan incubator shaker at 30° C. shaking at 250 rpm for 3 hours. The cellswere plated to determine the colony forming units (CFUs) that survivedelectroporation and failed to take up a plasmid and the CFUs thatsurvived electroporation and took up a plasmid. Plates were incubated at30° C.; E. coli colonies were counted after 24 hrs.

The flow-through electroporation experiments were benchmarked against 2mm electroporation cuvettes (Bull dog Bio) using an in vitro highvoltage electroporator (NEPAGENE™ ELEPO21). Stock tubes of cellsuspensions with DNA were prepared and used for side-to-side experimentswith the NEPAGENE™ and the flow-through electroporation. The results areshown in FIG. 22A. In FIG. 22A, the left-most bars hatched /// denotecell input, the bars to the left bars hatched \\\ denote the number ofcells that survived transformation, and the right bars hatched ///denote the number of cells that were actually transformed. The FTEPdevice showed equivalent transformation of electrocompetent E. colicells at various voltages as compared to the NEPAGENE™ electroporator.As can be seen, the transformation survival rate is at least 90% and insome embodiments is at least 95%, 96%, 97%, 98%, or 99%. The recoveryratio (the fraction of introduced cells which are successfullytransformed and recovered) is in certain embodiments at least 0.001 andpreferably between 0.00001 and 0.01. In FIG. 25A the recovery ratio isapproximately 0.0001.

Additionally, a comparison of the NEPAGENE™ ELEPO21 and the FTEP devicewas made for efficiencies of transformation (uptake), cutting, andediting. In FIG. 22B, triplicate experiments were performed where thebars hatched /// denote the number of cells input for transformation,and the bars hatched \\\ denote the number of cells that weretransformed (uptake), the number of cells where the genome of the cellswas cut by a nuclease transcribed and translated from a vectortransformed into the cells (cutting), and the number of cells whereediting was effected (cutting and repair using a nuclease transcribedand translated from a vector transformed into the cells, and using aguide RNA and a donor DNA sequence both of which were transcribed from avector transformed into the cells). Again, it can be seen that the FTEPshowed equivalent transformation, cutting, and editing efficiencies asthe NEPAGENE™ electroporator. The recovery rate in FIG. 22B for the FTEPis treater than 0.001.

For testing transformation of the FTEP device in yeast, S. Cerevisiaecells were created using the methods as generally set forth inBergkessel and Guthrie, Methods Enzymol., 529:311-20 (2013). Briefly,YFAP media was inoculated for overnight growth, with 3 ml inoculate toproduce 100 ml of cells. Every 100 ml of culture processed resulted inapproximately 1 ml of competent cells. Cells were incubated at 30° C. ina shaking incubator until they reached an OD₆₀₀ of 1.5+/−0.1.

A conditioning buffer was prepared using 100 mM lithium acetate, 10 mMdithiothreitol, and 50 mL of buffer for every 100 mL of cells grown andkept at room temperature. Cells were harvested in 250 ml bottles at 4300rpm for 3 minutes, and the supernatant removed. The cell pellets weresuspended in 100 ml of cold 1 M sorbitol, spun at 4300 rpm for 3 minutesand the supernatant once again removed. The cells were suspended inconditioning buffer, then the suspension transferred into an appropriateflask and shaken at 200 RPM and 30° C. for 30 minutes. The suspensionswere transferred to 50 ml conical vials and spun at 4300 rpm for 3minutes. The supernatant was removed and the pellet resuspended in cold1 M sorbitol. These steps were repeated three times for a total of threewash-spin-decant steps. The pellet was suspended in sorbitol to a finalOD of 150+/−20.

A comparative electroporation experiment was performed to determine theefficiency of transformation of the electrocompetent S. Cerevisiae usingthe FTEP device. The flow rate was controlled with a syringe pump(Harvard apparatus PHD ULTRA™ 4400). The suspension of cells with DNAwas loaded into a 1 mL glass syringe (Hamilton 81320 Syringe, PTFE LuerLock) before mounting on the pump. The output from the functiongenerator was turned on immediately after starting the flow. Theprocessed cells flowed directly into a tube with 1M sorbitol withcarbenicillin. Cells were collected until the same volume electroporatedin the NEPAGENE™ had been processed, at which point the flow and theoutput from the function generator were stopped. After a 3-hour recoveryin an incubator shaker at 30° C. and 250 rpm, cells were plated todetermine the colony forming units (CFUs) that survived electroporationand failed to take up a plasmid and the CFUs that survivedelectroporation and took up a plasmid. Plates were incubated at 30° C.Yeast colonies are counted after 48-76 hrs.

The flow-through electroporation experiments were benchmarked against 2mm electroporation cuvettes (Bull dog Bio) using an in vitro highvoltage electroporator (NEPAGENE™ ELEPO21). Stock tubes of cellsuspensions with DNA were prepared and used for side-to-side experimentswith the NEPAGENE™ and the flow-through electroporation. The results areshown in FIG. 23. The device showed better transformation and survivalof electrocompetent S. Cerevisiae at 2.5 kV voltages as compared to theNEPAGENE™ method. Input is total number of cells that were processed.

Example 4: Fully-Automated Singleplex RGN-directed Editing Run

Singleplex automated genomic editing using MAD7 nuclease wassuccessfully performed with an automated multi-module instrument of thedisclosure. See U.S. Pat. No. 9,982,279; and U.S. Ser. No. 16/024,831filed 30 Jun. 2018; Ser. No. 16/024,816 filed 30 Jun. 2018; Ser. No.16/147,353 filed 28 Sep. 2018; Ser. No. 16/147,865 filed 30 Sep. 2018;and Ser. No. 16/147,871 filed 30 Jun. 2018.

An ampR plasmid backbone and a lacZ_F172* editing cassette wereassembled via Gibson Assembly® into an “editing vector” in an isothermalnucleic acid assembly module included in the automated instrument.lacZ_F172 functionally knocks out the lacZ gene. “lacZ_F172*” indicatesthat the edit happens at the 172nd residue in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The assembled editing vector andrecombineering-ready, electrocompetent E. Coli cells were transferredinto a transformation module for electroporation. The cells and nucleicacids were combined and allowed to mix for 1 minute, and electroporationwas performed for 30 seconds. The parameters for the poring pulse were:voltage, 2400 V; length, 5 ms; interval, 50 ms; number of pulses, 1;polarity, +. The parameters for the transfer pulses were: Voltage, 150V; length, 50 ms; interval, 50 ms; number of pulses, 20; polarity, +/−.Following electroporation, the cells were transferred to a recoverymodule (another growth module), and allowed to recover in SOC mediumcontaining chloramphenicol. Carbenicillin was added to the medium after1 hour, and the cells were allowed to recover for another 2 hours. Afterrecovery, the cells were held at 4° C. until recovered by the user.

After the automated process and recovery, an aliquot of cells was platedon MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol and carbenicillin and grown until coloniesappeared. White colonies represented functionally edited cells, purplecolonies represented un-edited cells. All liquid transfers wereperformed by the automated liquid handling device of the automatedmulti-module cell processing instrument.

The result of the automated processing was that approximately 1.0 E⁻⁰³total cells were transformed (comparable to conventional benchtopresults), and the editing efficiency was 83.5%. The lacZ_172 edit in thewhite colonies was confirmed by sequencing of the edited region of thegenome of the cells. Further, steps of the automated cell processingwere observed remotely by webcam and text messages were sent to updatethe status of the automated processing procedure.

Example 5: Fully-Automated Recursive Editing Run

Recursive editing was successfully achieved using the automatedmulti-module cell processing system. An ampR plasmid backbone and alacZ_V10* editing cassette were assembled via Gibson Assembly® into an“editing vector” in an isothermal nucleic acid assembly module includedin the automated system. Similar to the lacZ_F172 edit, the lacZ_V10edit functionally knocks out the lacZ gene. “lacZ_V10” indicates thatthe edit happens at amino acid position 10 in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The first assembled editing vectorand the recombineering-ready electrocompetent E. Coli cells weretransferred into a transformation module for electroporation. The cellsand nucleic acids were combined and allowed to mix for 1 minute, andelectroporation was performed for 30 seconds. The parameters for theporing pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms;number of pulses, 1; polarity, +. The parameters for the transfer pulseswere: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses,20; polarity, +/−. Following electroporation, the cells were transferredto a recovery module (another growth module) allowed to recover in SOCmedium containing chloramphenicol. Carbenicillin was added to the mediumafter 1 hour, and the cells were grown for another 2 hours. The cellswere then transferred to a centrifuge module and a media exchange wasthen performed. Cells were resuspended in TB containing chloramphenicoland carbenicillin where the cells were grown to OD₆₀₀ of 2.7, thenconcentrated and rendered electrocompetent.

During cell growth, a second editing vector was prepared in theisothermal nucleic acid assembly module. The second editing vectorcomprised a kanamycin resistance gene, and the editing cassettecomprised a galK Y145* edit. If successful, the galK Y145* edit conferson the cells the ability to uptake and metabolize galactose. The editgenerated by the galK Y154* cassette introduces a stop codon at the154th amino acid reside, changing the tyrosine amino acid to a stopcodon. This edit makes the galK gene product non-functional and inhibitsthe cells from being able to metabolize galactose. Following assembly,the second editing vector product was de-salted in the isothermalnucleic acid assembly module using AMPure beads, washed with 80%ethanol, and eluted in buffer. The assembled second editing vector andthe electrocompetent E. Coli cells (that were transformed with andselected for the first editing vector) were transferred into atransformation module for electroporation, using the same parameters asdetailed above. Following electroporation, the cells were transferred toa recovery module (another growth module), allowed to recover in SOCmedium containing carbenicillin. After recovery, the cells were held at4° C. until retrieved, after which an aliquot of cells were plated on LBagar supplemented with chloramphenicol, and kanamycin. To quantify bothlacZ and galK edits, replica patch plates were generated on two mediatypes: 1) MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol, and kanamycin, and 2) MacConkey agar basesupplemented with galactose (as the sugar substrate), chloramphenicol,and kanamycin. All liquid transfers were performed by the automatedliquid handling device of the automated multi-module cell processingsystem.

In this recursive editing experiment, 41% of the colonies screened hadboth the lacZ and galK edits, the results of which were comparable tothe double editing efficiencies obtained using a “benchtop” or manualapproach.

Example 6: Design and Creation of a Yeast Display Library of PutativeTCR Antigens

The binding motifs for peptides presented by human MHC allele HLA-A*02have been well characterized (Falk, K., et al., Nature, 1991. 351(6324):p. 290-296; Glanville, J., et al., Nature, 2017. 547(7661): p. 94-98)and a number of restricted clinically relevant TCRs identified (Johnson,L. A., et al., Blood, 2009. 114(3): p. 535-546). A yeast-display libraryfor screening potential HLA-A*02:01 restricted TCRs is created asfollows. A library of approximately 10,000 oligonucleotide editingcassettes for introduction of synthetic pMHC (Glanville, J, supra)peptides of different sequence into the genome of S. Cerevisiae aredesigned and ordered from Agilent (Santa Clara, Calif.).

Briefly, the structural elements of each of the oligo cassettes is asfollows: a promoter region, a CRISPR guide RNA region, an optionalspacer region, a homology arm and optionally other sequences (e.g.,barcodes) helpful for further analysis based on the functional assay tobe used in the selection and/or confirmation of the specific edits. Thecassettes range in length from 180 nt to 230 nt, depending on the editto be introduced and the overall design of the oligos. The design of thehomology arm includes a synonymous codon change (if necessary) togenerate a restriction site which is used to insert constant regions ofthe cassette. These constant regions include the HLA-A*02:01 heavy chainand the AGA2P cell surface display conferring protein. The constantregion may also contain an epitope tag for ease of downstream use inselections.

Briefly, the structural elements of each of the oligo cassettes is asfollows: a promoter region, a CRISPR guide RNA region, an optionalspacer region, a homology arm and optionally other sequences (e.g.,barcodes) helpful for further analysis based on the functional assay tobe used in the selection and/or confirmation of the specific edits. Thecassettes range in length from 180 nt to 230 nt, depending on the editto be introduced and the overall design of the oligos. The design of thehomology arm includes a synonymous codon change (if necessary) togenerate a restriction site which is used to insert constant regions ofthe cassette. These constant regions include the HLA-A*02:01 heavy chainand the AGA2P cell surface display conferring protein. The constantregion may also contain an epitope tag or barcode “handle” for ease ofdownstream use in selections and further analysis. In addition oralternatively, the cassette design may include the addition of a“landing. pad” for the future addition of sequences. The CRISPR guideRNA region may also be targeted to a high efficiency cut and integrationsite.

Optionally, the oligonucleotide editing cassettes can be furtherprocessed with degenerate PCR reactions to generate 10⁷-10⁸ permutationsof the original TCR antigen sequence. Such degenerate PCR can beperformed either before or after introduction into the genome of thecells. Degenerate PCR reactions are performed with primers positionedover the portions of the intended edit representing the peptidedisplayed on the pMHC construct (See, e.g., Boder, E. T. and K. D.Wittrup, Nature Biotechnology, 1997. 15(6): p. 553-557; McMahon, C., etal., Nature Structural & Molecular Biology, 2018. 25(3): p. 289-296).

Importantly, combinatorial sequence diversity could be created anywherealong the heavy chain construct representing the HLA-A allele as well asin the peptide region. Individual yeast then express a random peptidetethered to the constant HLA molecule. HLA-A*02:01 typically presentspeptides 8 to 11 amino acids in length (Hassan, C., et al., The Journalof Biological Chemistry, 2015. 290(5): p. 2593-2603 and peptide lengthlibraries are generated using peptides of lengths within these ranges.The library has a theoretical nucleotide diversity dictated by thelibrary composition and length but is designed to result in one or morelibraries representing millions of unique peptides ranging from 8 to 11amino acids. After incubating the cells and going through the editingprocess, a pool of edited cells exists with the pMHC complex displayedon the surface of the cell attached to the AGA2P protein. An optionalinitial selection for edited cells displaying the pMHC complex can beperformed via the displayed epitope tag.

Example 7: Validation of the Proper Identification of TCR Antigens Usinga Yeast Display Library

A validation study is performed to determine whether the HLA-A*02:01complex on the surface of the cells in the library of Example 1 isproperly folded to present peptides. The validation uses theidentification of cells displaying target antigens of TCRs with knownspecificities. Briefly, a system is designed using the librariesgenerated as in Example 1 to validate the libraries for properexpression of the antigens. In this system, yeast cells displaying thepMHC conjugates are exposed to a population of expanded T-cells from asingle T-cell with known TCR. Using this system, a user can correctlymatch TCRs to a known predicted antigen target. Selections are performedusing TCRs with known antigen sequences. Following selection, theselected samples are determined, e.g. using sequencing of barcodesassociated with the selected antigens in the cells of the library. Thetop peptide antigens identified using the system of the disclosure areable to stimulate TCR-transduced T cells, despite sequence differencesfrom the actual epitope.

Example 8: Identification of New TCR Antigens Using a Yeast DisplayLibrary

To test whether the automated system would work to identify novelantigen targets, known TCRs and/or orphan TCRs are used to identifyantigens using the methods of the disclosure. These identified antigenscan then be used by bioinformatic methods to query the universe ofexpected or potential peptide antigens. These bioinformatics methodswill attempt to determine common peptides derived from known proteinsequences that will also bind the representative TCRs. These predictedpeptide sequences can then be designed into one of the libraries ofExample 1 or directly tested with other assays. These libraries whichare then displaying the predicted peptide pMHC molecules can then beexposed to one or more orphan TCRs to find antigens that specificallybind to the orphan TCRs. These peptides are then identified as probableantigen targets for the TCRs.

Example 9: Identification of Genome-Wide Protein-Protein Interactions

Protein-protein interactions have been traditionally studied inhigh-throughput using yeast two hybrid (Y2H) based approaches (Rolland,T., et al, Cell, 2014. 159(5): p. 1212-1226; Huttlin, E. L., et al.,2017. 545(7655): p. 505-509) or mass-spectrometry based assays (Hein,Marco Y., et al., Cell, 2015. 163(3): p. 712-723. Flow cytometry hasalso been used heavily to enable yeast surface display applications andhas been extended to facilitate studies of protein-protein interactionsand enzymatic properties (Lim, S., et al., Biotechnology Journal, 2017.12(5): p. 10; Cherf, G. M. and J. R. Cochran, Methods in molecularbiology (Clifton, N.J.), 2015. 1319: p. 155-175.

CREATE display can be used to facilitate rapid screening of one vs. allor all vs. all protein-protein interactions. First, a genome-wide CREATEdisplay library is generated by ordering a set of approximately 6,000oligonucleotide editing cassettes from Agilent (Santa Clara, Calif.).These oligonucleotide editing cassettes are configured as described inprevious examples with a crRNA, spacer region, and homology arm. Theseparticular oligonucleotide cassettes can also optionally containoptimally positioned restriction enzyme sites if they contain repetitivesequence to aid in the addition of a surface display conferring tag viastandard cloning methods. Many surface display conferring tags exist.McMahon, C., et al., supra; Cherf, G. M. and J. R. Cochran, Methods inmolecular biology (Clifton, N.J.), 2015. 1319: p. 155-175; Uchański, T.,et al., Scientific Reports, 2019. 9(1): p. 382. These may include andextend upon the original method of using the yeast AGA2P mating proteinthat is typically fused to the N-terminus of the displayed protein orpeptide of interest (Boder, E. T. and K. D. Wittrup, supra). Tofacilitate display of essential proteins critical to cellular function anon-optimal cleavage site could optionally be designed in-between thesurface display conferring tag and the protein of interest. Manycleavage conferring sequences exist but one exemplar is tobacco etchvirus (TEV) cleavage site which could be modified to result insub-optimal cleavage (Ioannou, M., et al., Mammalian expression vectorsfor metabolic biotinylation tandem affinity tagging by co-expression incis of a mammalian codon-optimized BirA biotin ligase. BMC researchnotes, 2018. 11(1): p. 390-390) and hence simultaneous surface displayof the desired protein while maintaining a viable intracellularconcentration of the native protein. Once oligonucleotide cassettes havebeen designed, ordered, and subsequently modified to include thestandard parts conferring display to the surface of the cell, the CREATEprocess can proceed. Briefly, as described previously, a population ofcells is transformed with the oligonucleotide cassettes and incubatedusing an automated machine that results in a population of edited cells.This population of cells is such that each individual cell contains oneor more edits that have resulted in insertion of the cell surfacedisplay conferring tag at a designed location of interest around thegenome. To create a genome-wide library displaying all proteins in theyeast genome approximately 6,572 edits would be made to insert surfacedisplay conferring tags at the N-terminus of all 6,572 annotatedproteins in the yeast genome(https://www.yeastgenome.org/genomesnapshot). This would result in alibrary of 6,572 distinct cells each displaying one of the 6,572proteins on its surface. This library of cells could then be split intotwo populations and one of the populations transformed with a constructexpressing green-fluorescent-protein (GFP). The two populations couldthen be incubated together and run through a flow-cytometer to detectdoublet formation (Wersto, R. P., et al., Cytometry, 2001. 46(5): p.296-306) indicative of a positive protein-protein interaction. Doubletscan then be placed into individual partitions of a standard 96 or 384well plate and the DNA sequence barcodes read off of the cassettespresent in each cell of the doublet to determine a protein-proteininteraction. Notably, this technique can be performed in an all-by-allmanner in which all 6,572 GFP expressing surface displayed proteins areincubated with all 6,572 non-GFP expressing surface displayed proteins.However, it can also be performed in a one-vs-all or many-vs-allconfiguration in which isolates of a protein of specific interest areincubated and sorted using flow cytometry as described above. Thisone-vs-all or many-vs-all could offer additional specificity or clarityto determination of an individual proteins binary interaction partners.It should also be noted that this same procedure can be done throughoutmultiple rounds of screening as is traditionally done in phage or yeastdisplay (Bradbury, A. R. M., et al., Nature Biotechnology, 2011. 29: p.245) to selectively enrich for the highest affinity binding partners andto lower false positive rates. It can also be used on a previouslyedited genome containing pathogenic or other variants of interest editedinto the cell population before introduction of the cell surface displayconferring tags. The previously edited genomes could also contain setsof variants specifically designed to disrupt protein-proteininteractions. Notably, CREATE display can also be used to display morethan one protein on the surface of a single cell via introduction ofmultiple tags at multiple loci throughout a cell.

Example 10: Identifying Druggable Targets

Identifying targets of drugs and subsequent mechanism of action remainsa challenging endeavor. Schenone, M., et al., Nature Chemical Biology,2013. 9: p. 232; Stockwell, B. R., Exploring biology with small organicmolecules. Nature, 2004. 432(7019): p. 846-854; Xie, L., L. Xie, and P.E. Bourne, Structure-based systems biology for analyzing off-targetbinding. Current opinion in structural biology, 2011. 21(2): p. 189-199.

Reverse genetic screens tend to use computational or other rationalmethods to pre-select a list of likely disease related targets.Biochemical screens are then performed using a library of chemicalcompounds against one or more of these disease related targets. However,biochemical assays are often costly or time consuming and subsequentlyare generally limited to a small number of potential targets. 17. Wyatt,P. G., et al., Target validation: linking target and chemical propertiesto desired product profile. Current topics in medicinal chemistry, 2011.11(10): p. 1275-1283.

The small number of feasible targets in biochemical screens oftentranslates into an inability to identify potential off-targets which canthen result in difficult to understand side effects and a necessary“deconvolution” step whilst determining mechanism of action. Knight, Z.A., H. Lin, and K. M. Shokat, Nature reviews. Cancer, 2010. 10(2): p.130-137.

In contrast, forward genetic screens generally start with a phenotype ofinterest and then screen a large number of molecules against the modelsystem to see if the phenotype can be disrupted. Stockwell, B. R.,Exploring biology with small organic molecules. Nature, 2004. 432(7019):p. 846-854.

This however can often result in not knowing what protein or pathway themolecule is targeting and can also lead to unintended side-effects whenadministered in further studies or in patients. Xie, L., et. Al., supra.

Both forward and reverse genetic screens could greatly benefit from theability to uniformly assess the binding of a drug to all intracellularproteins in a simple cost-effective assay. For forward screens it canidentify the actual targets and for reverse screens it can identifyoff-targets. Using the CREATE display methods described here, one canefficiently generate a library displaying all possible cellular proteinson the surface of a population of cells and then expose that populationto a small molecule with an attached fluorophore or other detectionhandle to determine all protein-drug binding events. First a CREATEdisplay library is generated as described in Example 5. This displaylibrary can optionally display all proteins in a genome or a subset ofproteins particular to a pathway or computationally determined set ofinterest. This display library can also be created in a population ofcells that already harbors one or more pathogenic variants identified apriori and programmed into the cell population via previous rounds ofCREATE. This library can then be exposed to a single molecule ofinterest with an attached organic fluorophore. Incubation of the CREATEdisplay library with the small molecule of interest results in complexesof small molecule bound to the cells displaying a protein in the case inwhich the small molecule can bind that protein. Using flow cytometry,the cells displaying protein with bound ligand can be sorted andbarcodes on the CREATE cassettes used to determine which proteins arebound by a given small molecule. This results in a binary mapping ofsmall molecule to protein and can uniquely identify all possible bindingpartners of a given small molecule. Optionally, using a DNA encodedchemical library or other combinatorial screening approaches(Zimmermann, G. and D. Neri, Drug discovery today, 2016. 21(11): p.1828-1834; Szymanski, P., M. Markowicz, and E. Mikiciuk-Olasik,International journal of molecular sciences, 2011. 13(1): p. 427-452)one could perform an all-by-all screen of a library of chemicalcompounds against a CREATE display library of surface displayedproteins.

Example 11: Affinity Maturation of Biological Binders to a Pathway ofInterest

Traditional antibody drug development has focused on cell surface orother extracellular targets that can be readily accessed by an antibody.However, of the approximately 700 protein molecular targets approved fordrugs, more than half are intracellular proteins. See, e.g., Carter, P.J. and G. A. Lazar, Nature Reviews Drug Discovery, 2017. 17: p. 197;Santos, R., et al., Drug discovery, 2017. 16(1): p. 19-34; Wang, X., etal., Genome biology and evolution, 2013. 5(7): p. 1291-1297.

Significant efforts are underway to develop delivery systems forantibodies or small peptide therapeutic molecules. Stewart, M. P., etal., Nature, 2016. 538: p. 183. If the promise of intracellular antibodyor peptide delivery comes to fruition, then a method to systematicallyaffinity mature antibodies that bind to one or more intracellularproteins would be of tremendous value. Using CREATE display, a largelibrary of intracellular proteins can be displayed on the surface of apopulation of cells and systematically exposed to yeast or phage displaylibraries to select for mono, bi, or poly-specific binders to a set oftargets. First, a yeast display library would be created via the methodsdescribed here or as described elsewhere (McMahon, C., et al., supra;Lim S. et al., supra; Cherf, G. M. and J. R. Cochran, supra) in whichmany combinatorically encoded proteins are encoded into a population ofyeast cells for display on the surface. At this point, the workflowwould proceed in the same fashion as laid out in Example 5. Inparticular, the library of cells with combinatorically encoded peptidesdisplayed on the surface would also be transformed with DNA sequencesconferring expression of green-fluorescent-protein. This library ofcells with up 10{circumflex over ( )}10 distinct displayed antibodies,nanobodies, or peptide fragments would then be incubated with the CREATEdisplayed library of all intracellular proteins. Using flow cytometryand selecting for doublets would then enable determination of anypairwise binding interaction between the engineered peptide(s) and oneor more surface displayed cellular proteins. This procedure could alsobe carried out iteratively in the same manner that traditional affinitymaturation of antibodies is done via yeast display (Cherf, G. M. and J.R. Cochran, supra). Carrying it out iteratively on a library of surfacedisplayed cellular proteins that represented a given pathway or subsetof genomic proteins would result in identification of high-affinitybinders to an entire pathway of proteins. In this manner poly-specificbinders could be determined to inhibit or identify the mechanism ofaction for entire pathways. Importantly, in a genome-wide CREATE displaylibrary there is a built-in negative control for off-target affects viathe presence in solution of all other intracellular proteins. Thus whileselecting for binders to only a subset of proteins in a pathway, one canfind the pareto optimum between strong binding to one or more desiredintracellular proteins while simultaneously minimizing binding tonon-desired intracellular proteins. Thus, throughout successive roundsof CREATE display one can affinity mature antibodies for binding tospecific targets while also selecting for minimization of off-targetbinding to other intracellular proteins.

While this invention is satisfied by embodiments in many differentforms, as described in detail in connection with embodiments of theinvention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific embodiments illustratedand described herein. Numerous variations may be made by persons skilledin the art without departure from the spirit of the invention. The scopeof the invention will be measured by the appended claims and theirequivalents. The abstract and the title are not to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, 916.

We claim:
 1. An automated method of creating a cell library expressingrandom peptides, the method comprising: processing a population of cellsusing an instrument for multiplexed nuclease-directed genome editingusing introduced nucleic acids and a nuclease, wherein the introducednucleic acids comprise nucleic acids that encode random peptidesconfigured to be displayed on surfaces of the cells, the random peptidescomprising unique polypeptides linked to immobilization peptides forimmobilization on the surfaces of the cells; incubating the processedcells to facilitate nucleic acid editing in the cells, wherein theedited cells express the random peptides in the cells; and allowing thecells to display the random peptides on the surfaces of the cells. 2.The method of claim 1, wherein the immobilization peptides comprise afirst binding motif that selectively binds to a second binding motifpresent on the surfaces of the cells.
 3. The method of claim 2, whereinthe first binding motif comprises avidin, streptavidin, or neutravidin.4. The method of claim 2, wherein the second binding motif comprisesbiotin.
 5. The method of claim 1, wherein the immobilization peptidescomprise a transmembrane polypeptide, a polypeptide membrane anchor, aGPI-linked polypeptide, or a natural surface polypeptide.
 6. The methodof claim 1, wherein the immobilization peptides are linked to aC-terminus or an N-terminus of the unique polypeptides.
 7. The method ofclaim 1, wherein the unique polypeptides comprise putative T-cellreceptor (TCR) antigens.
 8. The method of claim 1, wherein the uniquepolypeptides comprise predicted T-cell receptor (TCR) binding regions.9. The method of claim 1, wherein the random peptides are created byforward engineering.
 10. The method of claim 9, wherein the randompeptides are created by forward engineering based on predictions ofputative antigens useful for specific TCRs.
 11. An automated method ofcreating a cell library co-expressing engineered proteins and MHCmolecules, the method comprising: processing a population of cells usingan instrument for multiplexed nuclease-directed genome editing usingintroduced nucleic acids and a nuclease, wherein the introduced nucleicacids comprise nucleic acids that encode random peptides and MHCmolecules configured to be displayed on surfaces of the cells, therandom peptides and MHC molecules comprising unique polypeptides linkedto immobilization peptides for immobilization on the surfaces of thecells; incubating the processed cells to facilitate nucleic acid editingin the cells, wherein the edited cells co-express the random peptidesand MHC molecules in the cells; and allowing the cells to display therandom peptides and the MHC molecules on the surfaces of the cells. 12.The method of claim 11, wherein the immobilization peptides comprise afirst binding motif that selectively binds to a second binding motifpresent on the surfaces of the cells.
 13. The method of claim 12,wherein the first binding motif comprises avidin, streptavidin, orneutravidin.
 14. The method of claim 12, wherein the second bindingmotif comprises biotin.
 15. The method of claim 11, wherein theimmobilization peptides comprise a transmembrane polypeptide, apolypeptide membrane anchor, a GPI-linked polypeptide, or a naturalsurface polypeptide.
 16. The method of claim 11, wherein theimmobilization peptides are linked to a C-terminus or an N-terminus ofthe unique polypeptides.
 17. The method of claim 11, wherein theimmobilization peptides comprise a transmembrane polypeptide, apolypeptide membrane anchor, a GPI-linked polypeptide, or a naturalsurface polypeptide.
 18. The method of claim 11, wherein theimmobilization peptides are linked to a C-terminus or an N-terminus ofthe unique polypeptides.
 19. The method of claim 11, wherein the uniquepolypeptides comprise putative T-cell receptor (TCR) antigens.
 20. Themethod of claim 11, wherein the unique polypeptides comprise predictedT-cell receptor (TCR) binding regions.
 21. The method of claim 11,wherein the random peptides are created by forward engineering.
 22. Themethod of claim 21, wherein the random peptides are created by forwardengineering based on predictions of putative antigens useful forspecific TCRs.